Nanocapsule encapsulation system and method

ABSTRACT

The present invention generally relates to nanocapsules and methods of preparing these nanocapsules. The present invention includes a method of forming a surfactant micelle and dispersing the surfactant micelle into an aqueous composition having a hydrophilic polymer to form a stabilized dispersion of surfactant micelles. The method further includes mechanically forming droplets of the stabilized dispersion of surfactant micelles, precipitating the hydrophilic polymer to form precipitated nanocapsules, incubating the nanocapsules to reduce a diameter of the nanocapsules, and filtering or centrifuging the nanocapsules.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application is a continuation of application Ser. No.09/796,575 filed Feb. 28, 2001, which claims the benefit of U.S.Provisional Application No. 60/185,282 filed Feb. 28, 2000.

BACKGROUND OF THE INVENTION

[0002] The present invention generally relates to a field ofcontrolled-release delivery systems for macromolecules, particularlythose for nucleic acids and gene therapy. More specifically, the presentinvention relates to nanocapsules having a diameter of less than about50 nanometers, in which a bioactive component is located in a core ofthe nanocapsule, and to methods of forming these nanocapsules.

[0003] Over the past several decades, active and extensive research intothe use of nanoparticles in the delivery of bioactive agents hasgenerated a number approaches in the preparation of nanoparticles. Theseapproaches typically include the use of heat, high pressurehomogenization, or high intensity ultrasound sonication to preparenanoparticles having a diameter of more than 100 nanometers, or highamounts of solvents or oils, cytotoxic chemicals, such as cross-linkingagents, adjuvants, catalysts or any combination of any of these, toprepare nanoparticles having a diameter of less than 100 nanometers.Furthermore, these approaches are challenging due to a number ofvariables.

[0004] For example, when organic solvents are included in themanufacturing process for nanoparticles, the organic solvent maydenature the bioactive agent which reduces most, if not all, efficacy ofthe bioactive agent. In fact, denaturation of the bioactive agent maypromote a toxic response upon administration of the nanoparticle, to ahuman subject, for example.

[0005] In addition, when an organic solvent is used to preparenanoparticles, the organic solvent may undergo degradation to form a lowpH environment that destroys the efficacy of the bioactive agent.Therefore, organic

[0006] As a result, organic solvents are typically removed during themanufacturing process of nanoparticles. However, inclusion of one ormore organic solvent removal techniques generally increases the costsand complexity of forming nanoparticles.

[0007] The incorporation of high pressure homogenization or highintensity ultrasound sonication to prepare nanoparticles typicallyresults in entangling or embedding the bioactive agent in a polymericmatrix of the nanoparticle. Entangling or embedding the bioactive agentin the polymeric matrix may also denature the bioactive agent to therebyreduce the efficacy of the bioactive agent.

[0008] Entangling or embedding the bioactive agent in the polymericmatrix of the nanoparticle may also reduce the efficacy of the bioactiveagent by permitting premature release of the bioactive agent prior toreaching a target cell. Premature release of the bioactive agenttypically promotes cytotoxicity or cell death during administration ofthe nanoparticle.

[0009] Furthermore, nanoparticles that reach the target cell aretypically transported into the target cell via endosomal regulatedpathways that results in lysosomal degradation of the bioactive agentand the nanoparticle. Therefore, functional activity of the bioactiveagent inside the target cell may not occur since the bioactive agent andthe nanoparticle undergoes degradation. As used herein, the term“functional activity” refers to an ability of a bioactive agent tofunction within a target cell for purposes of providing a therapeuticeffect on the target cell.

[0010] Additionally, high pressure homogenization or high intensityultrasound sonication techniques often require complex and expensiveequipment that generally increases costs in preparing nanoparticles.Therefore, an urgent need exists to prepare nanoparticles without theuse of cytotoxic chemicals like organic solvents or the use of complexand expensive equipment. Furthermore, an urgent need exists to preparenanoparticles that do not entangle nor embed the bioactive agent in thenanoparticle so that cytotoxic responses are minimized. Additionally, anurgent need exists to develop a nanoparticle that maybe transported intoa target cell where the bioactive agent is released to accomplishtherapeutic delivery of the bioactive agent.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention generally relates to nanocapsules andmethods of preparing these nanocapsules. The present invention includesa method of forming a surfactant micelle and dispersing the surfactantmicelle into an aqueous composition having a hydrophilic polymer to forma stabilized dispersion of surfactant micelles. The method furtherincludes mechanically forming droplets of the stabilized dispersion ofsurfactant micelles, precipitating the hydrophilic polymer to formprecipitated nanocapsules, incubating the nanocapsules to reduce adiameter of the nanocapsules, and filtering or centrifuging thenanocapsules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a schematic of a method of the present invention forpreparing nanocapsules.

[0013]FIG. 1A illustrates atomic force microscopy of nanocapsuleformulations prepared under different dispersion conditions.

[0014]FIG. 1B illustrates results from an experiment documentingquantitative recovery of small amounts of DNA from releasing solutions.

[0015]FIG. 1C illustrates cumulative release over 72 hours fornanocapsules prepared under different dispersion conditions.

[0016]FIG. 2 illustrates relative pinocytotic activity of HacaTkeratinocyte cultures treated with DNA complexes, nanocapsulescontaining DNA or no treatment.

[0017]FIG. 3 illustrates the results of western blotting of totalprotein from rat fibroblast cultures.

[0018]FIG. 4A illustrates

[0019]FIG. 4B illustrates immunofluorescence microscopy of porcinedermal tissue sections from the experiment of FIG. 4.

[0020]FIG. 5 shows incorporation of nanocapsules into a solid dosageform.

[0021]FIG. 6A illustrates polyvinylpyrolidone nanocapsule uptake andGreen Fluorescent Protein (GFP) expression in 35 mm human dermalfibroblast and immortalized keratinocyte cultures.

[0022]FIG. 6B illustrates tumor targeting of GFP plasmid DNA by Tenascinnanocapsules.

[0023]FIG. 6C illustrates an effect of nanocapsules that are coated withTenascin and nanocapsules that are not coated with Tenascin on growthinhibition of squamous cell carcinoma and human dermal fibroblast (HDF)cultures.

[0024]FIG. 7 shows uptake of HDF cultures treated with nanocapsulescontaining 20 mer Fitc-labeled O-methyl RNA oligonucleotides.

DETAILED DESCRIPTION

[0025] The present invention generally relates to nanocapsules having adiameter of less than about 50 nanometers (nm). The present inventionalso relates to a method of preparing these nanocapsules. According tothe method of the present invention, a nanocapsule is formed bypartitioning a bioactive component within a core of surfactantmolecules, and surrounding the surfactant molecules with a biocompatiblepolymer shell.

[0026] A method for producing the nanocapsule is generally depicted at10 in FIG. 1. In the method 10, a bioactive component 12 ishomogeneously dispersed into a first aqueous composition 14 to form ahydrophilic composition (not shown). Next, a surfactant composition 16,including a surfactant component (not shown) that contains a pluralityof surfactant molecules, and an optional biocompatible oil component 18,are introduced into a first dispersing apparatus 20 along with thehydrophilic composition. The surfactant composition 16 is subjected toconditions in the first dispersing apparatus 20 that initiate at leastpartial adsorption of the surfactant molecules onto a surface of thebioactive component 12.

[0027] Partial adsorption of surfactant molecules onto the surface ofthe bioactive component 12 initiates partitioning of the bioactivecomponent 12 into a core of a shell formed from the surfactant moleculesin the first aqueous composition 14. Adsorption of the surfactantmolecules onto the surface of the bioactive component 12 may proceeduntil an entire surface of the bioactive component 12 is covered by thesurfactant molecules to complete partitioning of the bioactive component12 into the core of surfactant molecules and form a surfactant micelle22.

[0028] Next, a biocompatible polymer component 24 is added to thesurfactant micelle 22 to stabilize the surfactant micelle 22 located inthe first aqueous composition 14. Preferably, the biocompatible polymercomponent 24 surrounds the surfactant micelle 22 in a stabilizingapparatus 26 to form a stabilized surfactant micelle 28.

[0029] After stabilization, the stabilized surfactant micelle 28 istransferred from the stabilizing apparatus 26 into a second aqueouscomposition 30 located in a second dispersing apparatus 32. Preferably,the second aqueous composition 30 includes a solute (not shown) that iscapable of precipitating the biocompatible polymer component 24 thatcoats the stabilized surfactant micelle 28. After precipitating thebiocompatible polymer component 24 of the stabilized surfactant micelle28, dispersed, optionally atomized precipitated nanocapsules 36,hereinafter referred to as nanocapsules 36, are formed.

[0030] It has been discovered that dispersing a surfactant composition,that includes a surfactant component having ahydrophile-lipophile-balance (HLB) value of less than about 6.0 units,into an aqueous composition that contains a bioactive component formssurfactant micelles that surround the bioactive component. It hasfurther been discovered that stabilizing the surfactant micelles byadding a biocompatible polymer coats the surfactant micelles to formnanocapsules having a diameter of less than about 50 nm.

[0031] As used herein, the term “nanoparticle” refers a particle havinga matrix-type structure with a size of less than about 1,000 nanometers.When the nanoparticle includes a bioactive component, the bioactivecomponent is entangled or embedded in the matrix-type structure of thenanoparticle.

[0032] The term “nanosphere”, as used herein, refers to a particlehaving a solid spherical-type structure with a size of less than about1,000 nanometers. When the nanosphere includes a bioactive component,the bioactive component is adsorbed onto the surface of the nanosphereor embedded in the nanosphere.

[0033] Similarly, the term “nanocore”, as used herein, refers to aparticle having a solid core with a size of less than about 1,000nanometers. When the nanocore includes a bioactive component, thebioactive component is entangled in the nanocore.

[0034] As used herein, the term “nanocapsule” refers to a particlehaving a hollow core that is surrounded by a shell, such that theparticle has a size of less than about 1,000 nanometers. When ananocapsule includes a bioactive component, the bioactive component islocated in the core that is surrounded by the shell of the nanocapsule.The term “nanocapsule” is not meant to encompass, and generally does notinclude, a particle having a size of less than about 1,000 nanometers,in which a bioactive component is entangled or embedded in the matrix ofthe nanocapsule or adsorbed onto the surrounding shell of thenanocapsule.

[0035] The bioactive component 12 may be included into the first aqueouscomposition 14 as a liquid, vapor or in granular form. The form of thebioactive component 12 that is selected preferably permits the bioactivecomponent 12 to (1) remain stable prior to dissolving or dispersing intothe first aqueous composition 14, (2) be homogeneously dispersed intothe first aqueous composition 14, (3) be optionally condensed to reducea size of the bioactive component 12, (4) be partitioned into the coreof the surfactant micelles 22, (5) be released upon degradation of thebiocompatible polymer shell 24 of the nanocapsule 36, and (6) befunctionally active upon release from the nanocapsule 36.

[0036] The bioactive component 12 may be characterized as “hydrophilic”or “hydrophobic”. As used herein, the term “hydrophilic” and“hydrophilicity” refers to an ability of a molecule to adsorb water orform one or more hydrogen-bond(s) with water. All references to“hydrophilic” is also understood as encompassing any portion of themolecule that is capable of adsorbing water or forming one or morehydrogen-bond(s) with water. As used herein, the term “hydrophobic” and“hydrophobicity” refers to an ability of a molecule to not adsorb waternor form one or more hydrogen-bond(s) with water. All references to“hydrophobic” is also understood as encompassing any portion of themolecule that is not capable of adsorbing water nor forming one or morehydrogen-bond(s) with water.

[0037] When the bioactive component 12 is a hydrophilic bioactivecomponent, the hydrophilic bioactive component maybe directly added tothe first aqueous composition 14. As an alternative, the hydrophilicbioactive component 12 may be optionally dissolved or dispersed in oneor more solvents, such as water, a nonpolar solvent, a polar solvent, orany combination of any of these.

[0038] As used herein, the term “nonpolar solvent” refers to a solventthat does not have a permanent electric dipole moment, and therefore hasno ability for an intramolecular association with a polar solvent.Additionally, a nonpolar solvent may be characterized as a solvent thatincludes molecules having a dielectric constant of less than about 20units. Similarly, the term “immiscible”, as used herein, refers to aninability of two or more substances, such as two or more liquids,solids, vapors, or any combination of any of these, to form anintramolecular association with another substance. Some non-exhaustiveexamples of nonpolar solvents may be found in Perry's ChemicalEngineer's Handbook, Sixth Edition, which is incorporated herein byreference.

[0039] As used herein, the term “polar solvent” refers to a solvent thathas a permanent electrical dipole moment, and therefore has an abilityto form an intramolecular association with another polar substance, suchas a liquid, a solid, a vapor or any combination of any of these.Additionally, a polar solvent may be characterized as a solvent thatincludes molecules having a dielectric constant of more than about 20units. Likewise, the term “miscible”, as used herein, refers to anability of two or more substances to form an intramolecular associationwith each other. Some non-exhaustive examples of polar solvents may befound in Perry's Chemical Engineer's Handbook, Sixth Edition, which hasbeen incorporated herein by reference.

[0040] When the bioactive component 12 is a hydrophobic bioactivecomponent, the hydrophobic bioactive component may be dispersed ordissolved in a solvent that is capable of dispersing or dissolving thehydrophobic molecule, such as the above-mentioned water, a nonpolarsolvent, a polar solvent, or any combination of any of these.Preferably, when the bioactive component 12 is a hydrophobic bioactivecomponent 12, the hydrophobic bioactive component 12 is dissolved ordispersed in a water-miscible solvent, such as, acetone, acetonitrile,ethanol, dimethyl acetamide (DMA), tetrahydrofuran (THF), dioxane,dimethylsulfoxide (DMSO), and dimethylformamide (DMF). Other suitablenon-exhaustive examples of water-miscible solvents may be found inPerry's Chemical Engineer's Handbook, Sixth Edition, which has beenincorporated herein by reference.

[0041] As noted, the bioactive component 12 may be optionally condensedin the first aqueous composition 14 prior to forming the surfactantmicelle 16. For example, when the bioactive component is apolynucleotide, the polynucleotide may be condensed using aDNA-condensing agent. As used herein, a “DNA-Condensing Agent” is amolecule that facilitates condensation or a size reduction of DNA.

[0042] While condensation of the bioactive component 12 is not criticalto the present invention, condensation of the bioactive component 12maybe practiced to reduce the size of the bioactive component 12.Condensation of the bioactive component 12 generally reduces the size ofthe bioactive component 12 prior to partitioning into the core of thesurfactant micelle 16. Reducing the size of the bioactive component 12may permit maximum incorporation of the bioactive component 12 into thesurfactant micelle 22 or may assist a reduction in the overall size ofthe nanocapsule 36. Increasing the amount of the bioactive component 12that may be included as part of the nanocapsule 36 permits incorporationof macromolecules having a large number of monomers, such as a largenumber of base pairs or amino acids, for example. Some non-exhaustiveexamples of condensing agents have been reviewed in Rolland, A. P.(1998). Crit. Rev. Therapeutic Drug. Carr. Syst. 15:143-198, and isincorporated herein by reference.

[0043] The bioactive component 12 may further include additionalcomponents that are compatible with, and that do not interfere withsolvation or dispersion of the bioactive component 12. Somenon-exhaustive examples of additional components that may be added tothe bioactive component 12 include a DNA-associating moiety, whichrefers to a molecule, or portions thereof, that interact in anon-covalent fashion with nucleic acids. DNA-associating moieties mayinclude, but are not limited to, a major-and minor-groove binder, a DNAintercalator, a polycation, a DNA-masking component, amembrane-permeabilizing component, a subcellular-localization component,or the like. Major- and minor-groove binders, as used herein, aremolecules thought to interact with DNA by associating with the major orminor groove of double-stranded DNA.

[0044] Similarly, the term “DNA intercalator”, as used herein, refer toa planar molecule or planar portion of a molecule thought to intercalateinto DNA by inserting themselves between, and parallel to, a nucleotidebase pair. As used herein, a “polycation” is thought to associate withthe negative charges on the DNA backbone. The DNA-associating moiety maybe covalently linked through a “reactive group” to a functionalcomponent of this invention. The reactive group is easily reacted with anucleophile on the functional component. Some non-exhaustive examples ofreactive groups (with their corresponding reactive nucleophiles)include, but are not limited to N-hydroxysuccinimide (e.g., amine),maleimide and maleimidophenyl (e.g., sulfhydryl), pyridyl disulfide(e.g., sulfhydryl), hydrazide (e.g., carbohydrate), and phenylglyoxal(e.g., arginine).

[0045] The term “DNA-masking component”, as used herein, refers to amolecule capable of masking all or part of a polynucleotide followingrelease from a nanocapsule to increase its circulatory half-life byinhibiting attack by degrading reagents, such as nucleases, present inthe circulation and/or interfering with uptake by thereticuloendothelial system. Similarly, the term “membrane-permeabilizingcomponent”, as used herein, refers to any component that aids in thepassage of a polynucleotide or encapsulated polynucleotide across amembrane. Therefore, “membrane permeabilizing component” encompasses inpart a charge-neutralizing component, usually a polycation, thatneutralizes the large negative charge on a polynucleotide, and enablesthe polynucleotide to traverse the hydrophobic interior of a membrane.

[0046] Many charge-neutralizing components can act asmembrane-permeabilizers. Membrane-permeabilization may also arise fromamphipathic molecules. A “membrane permeabilizer”, as used herein, is amolecule that can assist a normally impermeable molecule to traverse acellular membrane and gain entrance to the cytoplasm of the cell. Themembrane permeabilizer may be a peptide, bile salt, glycolipid,phospholipid or detergent molecule. Membrane permeabilizers often haveamphipathic properties such that one portion is hydrophobic and anotheris hydrophilic, permitting them to interact with membranes.

[0047] The term “subcellular-localization component”, as used herein,refers to a molecule capable of recognizing a subcellular component in atargeted cell. Recognized subcellular components include the nucleus,ribosomes, mitochondria, and chloroplasts. Particularsubcellular-localization components include the “nuclear-localizationcomponents” that aid in carrying molecules into the nucleus and areknown to include the nuclear localization peptides and amino acidsequences.

[0048] The bioactive component 12 may be included at an amount that istherapeutically effective to transform a plurality of cells, such as invitro, in vivo or ex vivo cells. As used herein, “transform” refers to apresence and/or functional activity of the bioactive component in theplurality of cells after exposing the nanocapsules to the plurality ofcells.

[0049] Furthermore, those of ordinary skill in the art will recognizethat the amount of the bioactive component 12 may vary depending uponthe bioactive component 12, the temperature, pH, osmolarity, anysolutes, any additional component or optional solvents present in thesecond aqueous component 30, the surfactant composition 16, a type or anamount of the surfactant micelle 22, the biocompatible polymer component24, any desired characteristics of the stabilized surfactant micelle 28,any desired characteristics of the nanocapsules 36, or any combinationof any of these.

[0050] The bioactive component 12 of the nanocapsule 36 maybe suppliedas an individual macromolecule or supplied in various prepared mixturesof two or more macromolecules that are subsequently combined to form thebioactive component 12. Some non-exhaustive examples of hydrophilicmacromolecules that may be suitable for inclusion as part of thebioactive component 12 include, but are not limited to polynucleotides,polypeptides, genetic material, peptide nucleic acids, aptamers,carbohydrates, mini-chromosomes, molecular polymers, aggregates orassociations of an inorganic or organic nature, genes, any otherhydrophilic macromolecule or any combination of any of these.

[0051] Some non-exhaustive examples of hydrophobic macromolecules thatmay be included part of the bioactive component 12 include, but are notlimited to, adregergic, adrenocotical steroid, adrenocorticalsuppressant, aldosterone antagonist, and anabolic agents; analeptic,analgesic, anesthetic, anorectic, and anti-acne agents; anti-adrenergic,anti-allergic, anti-amebic, anti-anemic, and anti-anginal agents;anti-arthritic, anti-asthmatic, anti-atherosclerotic, antibacterial, andanticholinergic agents; anticoagulant, anticonvulsant, antidepressant,antidiabetic, and antidiarrheal agents; antidiuretic, anti-emetic,anti-epileptic, antifibrionlytic, and antifungal agent; antihemorrhagic,inflammatory, antimicrobial, antimigraine, and antimiotic agents;antimycotic, antinauseant, antineoplastic, antineutropenic, andantiparasitic agents; antiproliferative, antipsychotic, antirheumatic,antiseborrheic, and antisecretory agents; antipasmodic, antihrombotic,antiulcerative, antiviral, and appetite suppressant agents; bloodglucose regulator, bone resorption inhibitor, bronchodilator,cardiovascular, and cholinergic agents; fluorescent, free oxygen radicalscavenger, gastrointestinal motility effector, glucocorticoid, and hairgrowth stimulant agent; hemostatic, histamine H2 receptor antagonists;hormone; hypocholesterolemic, and hypoglycemic agents; hypolipidemic,hypotensive, and imaging agents, immunizing and agonist agents; moodregulators, mucolytic, mydriatic, or nasal decongestant; neuromuscularblocking agents; neuroprotective, NMDA antagonist, non-hormonal sterolderivative, plasminogen activator, and platelet activating factorantagonist agent; platelet aggregation inhibitor, psychotropic,radioactive, scabicide, and sclerosing agents; sedative,sedative-hypnotic, selective adenosine A1 antagonist, serotoninantagonist, and serotonin inhibitor agent; serotonin receptorantagonist, steroid, thyroid hormone, thyroid hormone, and thyroidinhibitor agent; thyromimetic, tranquilizer, amyotrophic lateralsclerosis, cerebral ischemia, and Paget's disease agent; unstableangina, vasoconstrictor, vasodilator, wound healing, and xanthineoxidase inhibitor agent; immunological agents, antigens from pathogens,such as viruses, bacteria, fungi and parasites, optionally in the formof whole inactivated organisms, peptides, proteins, glycoproteins,carbohydrates, or combinations thereof, any examples of pharmacologicalor immunological agents that fall within the above-mentioned categoriesand that have been approved for human use that may be found in thepublished literature, any other hydrophobic bioactive component, or anycombination of any of these.

[0052] As used herein, the term “polypeptide” refers to a polymer ofamino acids not limited by the number of amino acids. It is also to beunderstood that the term “polypeptide” is meant to encompass anoligopeptide, a peptide, or a protein, for example.

[0053] As used herein, the term “polynucleotide” refers to RNA or DNAsequences of more than 1 nucleotide in either single chain, duplex ormultiple chain form. The term “polynucleotide” is also meant toencompass polydeoxyribonucleotides containing 2′-deoxy-D-ribose ormodified forms thereof, RNA and any other type of polynucleotide whichis an N-glycoside or C-glycoside of a purine or pyrimidine base, ormodified purine or pyrimidine base or basic nucleotide. Thepolynucleotide may encode promoter regions, operator regions, structuralregions, termination regions, combinations thereof or any othergenetically relevant material. Similarly, the term “genetic” as usedherein, refers to any material capable of modifying gene expression.

[0054] The first aqueous composition 14 may be included in the method ofthe present invention as a gel, liquid, or in vapor form. The form ofthe first aqueous composition 14 that is selected preferably permits thefirst aqueous composition 14 to (1) remain stable prior to dissolving ordispersing the bioactive component, the surfactant composition 16, thesurfactant micelle 22, or optionally the stabilizer surfactant micelle28, (2) homogeneously disperse the bioactive component 12, thesurfactant composition 16, the surfactant micelle 22, or optionally thestabilizer surfactant 28, (3) function as a continuous phase in anoil-in-water emulsion, (4) not interfere with, or mask the functionalactivity of the bioactive component 12, and (5) not modify or degradethe bioactive component 12, the surfactant composition 16, thesurfactant micelle 22, or optionally the stabilized surfactant micelle28.

[0055] The first aqueous composition 14 may include only water, or mayoptionally include additional solutes or solvents that do not interferewith the method of forming the nanocapsules 36 nor mask the functionalactivity of the bioactive component 12. Furthermore, those of ordinaryskill in the art will recognize that an amount of the first aqueouscomposition 14 used to prepare the nanocapsules 36 may vary dependingupon the bioactive component 12, the surfactant composition 16, thetemperature, pH, osmolarity, optional solutes or optional solvents, thesurfactant micelle 22, the biocompatible polymer component 24, anydesired characteristics of the stabilized surfactant micelle 28 or thenanocapsules 36.

[0056] The bioactive component 12 may be added to the first aqueouscomposition 14 or the first aqueous composition 14 may be added to thebioactive component 12. While the order of addition of the bioactivecomponent 12 and the first aqueous composition 14 is not critical to thepresent invention, the hydrophilic composition (not shown) that isformed when the bioactive component 12 is dissolved or dispersed in thefirst aqueous composition 14 is preferably capable of maintaining ahomogeneous solution or dispersion in the hydrophilic composition.

[0057] The first aqueous composition 14 may be supplied as an individualcomponent or supplied in various prepared mixtures of two or morecomponents that are subsequently combined to form the first aqueouscomposition 14. Some non-exhaustive examples of the first aqueouscomposition 14 include, but are not limited to, the above-mentionedwater, nonpolar solvents, polar solvents, or any combination of any ofthese. Preferably, water is the first aqueous composition 14.

[0058] The surfactant composition 16 maybe introduced into the bioactivecomponent 12, the first aqueous composition 14, the hydrophiliccomposition as a liquid, vapor or in granular form. The form of thesurfactant composition 16 that is selected preferably permits thesurfactant composition 16 to (1) remain stable prior to introducing intothe bioactive component 12, the first aqueous composition 14, or thehydrophilic composition, (2) be homogeneously dispersed into thebioactive component 12, the first aqueous composition 14, or thehydrophilic composition, (3) form a micellar structure, (4) be adsorbedonto a surface of the bioactive component 12, the first aqueouscomposition 14, the hydrophilic composition (5) displace the firstaqueous composition that is located on the surface of the bioactivecomponent 12, (6) partition the bioactive component 12 or thehydrophilic composition into a core of the micellar structure to formthe surfactant micelle 22, and (7) provide a thermodynamic driving forcethat is effective to reduce a size of the bioactive component 12,surfactant micelle 22, the stabilized surfactant 28 or the nanocapsule36.

[0059] As used herein, a “surfactant” refers to any molecule containinga polar portion that thermodynamically prefers to be solvated by a polarsolvent, and a hydrocarbon portion that thermodynamically prefers to besolvated by a non-polar solvent. The term “surfactant” is also meant toencompass anionic, cationic, or non-ionic surfactants. As used herein,the term “anionic surfactant” refers to a surfactant with a polarportion that ionizes to form an anion in aqueous solution. Similarly, a“cationic surfactant” refers to a surfactant having a cationic polarportion that ionizes to form a cation in aqueous solution. Likewise, a“non-ionic” surfactant refers to a surfactant having a polar portionthat does not ionize in aqueous solution.

[0060] While not wanting to be bound to theory, it is generally believedthat a surfactant refers to a molecule that is effective to reduce asurface or an interfacial tension between a first substance dispersed ina second substance such that the first substance is solvated and anymolecular groups of the first substance are dispersed. Typically, ahydrodynamic diameter of the first substance increases after addition ofthe surfactant. Nonetheless, the surfactant composition 16 is believedto be effective to reduce the size or diameter of the surfactantmicelles 22 in the first aqueous composition 14, to thereby reduce thesize of the nanocapsule 36 when practicing the present invention.

[0061] The surfactant composition 16 may include the surfactantcomponent only (not shown), or may optionally include the biocompatibleoil component 18. The surfactant component may be characterized on theHLB scale that ranges from less than about 1 to more than about 13units.

[0062] A surfactant component having an HLB value of less than about 6.0units may be described as being poorly, or not dispersable in an aqueousor water-based composition. In addition, a surfactant component havingan HLB value of less than about 6.0 units may be characterized as ahydrophobic or non-ionic surfactant. A surfactant component having anHLB value of more than about 7.0 units may be described as being capableof forming a milky to translucent to clear dispersion when thesurfactant having an HLB value of more than about 7.0 units is dispersedin an aqueous or water-based composition.

[0063] Preferably, the surfactant component of the surfactantcomposition 16 has an HLB value of less than about 6.0 units whenpracticing the method of the present invention. Still more preferablythe surfactant component of the surfactant composition 16 has an HLBvalue of less than about 5.0 units to facilitate preparation ofnanocapsules having a diameter of less than about 50 nm.

[0064] The surfactant component may also be characterized in terms of acritical micelle concentration (CMC) value. Preferably, the surfactantcomponent of the surfactant composition 16 has a CMC value of less thanabout 300 micromolars (μm). Still more preferably, the surfactantcomponent has a CMC value of less than about 200 μm.

[0065] While not wanting to be bound to theory, it is believed that thesurfactant component of the surfactant composition 16 adsorbs onto thesurface of the bioactive component 12 when introduced into the firstaqueous composition 14 to minimize exposure of a surface of thehydrophobic surfactant component to a thermodynamically unfavorableenvironment created by the first aqueous composition 14. Therefore, thesurfactant component adsorbs onto the surface of the bioactive componentto reduce the surface area of the surfactant component that may beexposed to the first aqueous composition 14. Adsorption of thesurfactant component onto the bioactive component 12 is believed tofacilitate the size reduction of the bioactive component 12 and/or thesurfactant micelle 22.

[0066] The surfactant component of the surfactant composition 16 may besupplied as individual surfactants or supplied in various preparedmixtures of two or more surfactants that are subsequently combined toform the surfactant composition 16. Some non-exhaustive examples ofsuitable surfactants having an HLB value of less than about 6.0 units ora CMC value of less than about 200 μm be listed in DermatologicalFormulations (Barry, B., Marcel Dekker, (1983)), or in Percutaneousabsorption: drug, cosmetics, mechanisms, methodology, 3^(rd) ed.,Bronough, R. ed., 1999, or the Handbook of Industrial Surfactants (Ash,M., Ed., Gower Pub. (1993), which are incorporated herein by reference.As an example, the surfactant component maybe 2, 4, 7,9-tetramethyl-5-decyn-4, 7-diol(TM-diol), blends of 2, 4, 7,9-tetramethyl-5-decyn-4, 7-diol(TM-diol), molecules having one or moreacetylenic diol groups, cetyl alcohol or any combination of any ofthese.

[0067] The optional biocompatible oil component 18 of the surfactantcomposition 16 may be combined with the surfactant component as aliquid, vapor or in granular form. The form of the optionalbiocompatible oil component 18 that is selected preferably permits theoptional biocompatible oil component 18 to (1) remain stable prior tointroduction into the surfactant composition 16, (2) be homogeneouslyblended into the surfactant composition 16, (3) dissolve or disperse thesurfactant component, and (4) increase the hydrophobicity of thesurfactant composition 16, and therefore, the degree to which the sizeof the bioactive component 12, the surfactant micelle 22, the stabilizersurfactant micelle 28, or the nanocapsule 36 may be reduced whenpracticing the present invention.

[0068] Preferably, the concentration of the optional biocompatible oilcomponent 18 in the surfactant composition 16 ranges from about 10⁻⁷weight percent to about 10 weight percent, based upon a total volume ofthe stabilized surfactant micelles 28 in the first aqueous composition14. Concentrations of the optional biocompatible oil component 18 higherthan about 10 weight percent, based upon the total volume of thesurfactant composition 18, may be less desirable because such higherconcentrations increase a phase volume of the biocompatible oil, andconsequently may cause difficulties in preparing, dispersing and/orhandling the surfactant micelles 22, the stabilized surfactant micelles28 or the nanocapsules 36. Concentrations of the optional biocompatibleoil component lower than about 10⁻⁷ weight percent in the surfactantcomposition 16 may be less preferred, because such lower concentrationswould not be effective to solvate the surfactant component, or increasethe hydrophobicity of the surfactant composition 16, and may ultimatelyincrease the diameter of the nanocapsules 36.

[0069] The optional biocompatible oil component 18 of the surfactantcomposition 16 may be supplied as an individual biocompatible oil orsupplied in various prepared mixtures of two or more biocompatible oilsthat are subsequently combined to form the optional biocompatible oilcomponent 18. Some non-exhaustive examples of suitable biocompatibleoils that may be included as part of the biocompatible oil component 18may be found in Dermatological Formulations (Barry, B., Marcel Dekker,(1983)), or in Percutaneous absorption: drug, cosmetics, mechanisms,methodology, 3^(rd) ed., Bronough, R. ed. , 1999, or in the Handbook ofIndustrial Surfactants (Ash, M., Ed., Gower Pub. (1993), which have beenincorporated herein by reference. Preferably, food or USP grade oils,such as DMSO, DMF, castor oil, or any combination thereof, are used topractice the present method.

[0070] The surfactant composition 16 may be included at an amount thatis effective to form the micellar structure that partitions thebioactive component 12, the first aqueous composition 14 or thehydrophilic composition into the core of the micellar structure whenforming the surfactant micelle 22. Still more preferably, the surfactantcomposition 16 is included at an amount that is effective to provide amaximum thermodynamic driving force that minimizes the size of thebioactive component 12, the surfactant micelle 22, and ultimately, thesize of the nanocapsule 36 when practicing the present invention.

[0071] Furthermore, those of ordinary skill in the art will recognizethat the amount of the surfactant composition 16 may be varied basedupon the bioactive component 12, the first aqueous composition 14, aratio of the surfactant component to the optional biocompatible oil 18,any desired characteristics of the surfactant micelles 22, thestabilized surfactant micelles 28 or the nanocapsules 36. For example, asurfactant composition containing a surfactant component having an HLBvalue of about 6.0 units mixed with a nonpolar biocompatible oil likecastor oil, may provide the same degree of a thermodynamic driving forceas a second surfactant composition containing a surfactant component ofabout 4.0 units mixed with DMSO.

[0072] The amount of the surfactant composition 16 may range up to about0.5 weight percent, based upon a total volume of the stabilizedsurfactant micelles dispersed in the first aqueous composition 14. Stillmore preferably, the amount of the surfactant composition 16 is lessthan about 0.25 weight percent, based upon the total volume of thestabilized surfactant micelles 28 dispersed in the first aqueouscomposition 14. Most preferably, the surfactant composition 16 ispresent at an amount of less than about 0.05 weight percent, based uponthe total volume of the stabilized surfactant micelles 28 dispersed inthe first aqueous composition 14. As one non-exhaustive example, thesurfactant composition 16 may be added to the total volume of thehydrophilic composition at a concentration of about 500 ppm, based onthe total volume of the stabilized surfactant micelles 28 in the firstaqueous composition 14.

[0073] The first dispersing apparatus 20 initiates and promotesformation of the micellar structures that are based on the bioactivecomponent 12, the first aqueous composition 14 and the surfactantcomposition 16. Adsorption of surfactant component onto the surface ofthe bioactive component 12, or hydrophilic composition continues untilall of the surfactant molecules cover, and therefore, entrap thebioactive component 12 or hydrophilic composition in the core of themicellar structure to form surfactant micelles 22. Formation of aplurality of surfactant micelles 22 in the first aqueous composition 14forms a dispersion of surfactant micelles 22.

[0074] In general, any conventional dispersing apparatus 20 that iscapable of homogenously blending or dispersing may be suitable for usein forming the dispersion of surfactant micelles in accordance with thepresent invention. Furthermore, those of ordinary skill in the art willrecognize that the first dispersing apparatus 20 may vary depending uponthe desired characteristics of the nanocapsules 36. For example, thefirst dispersing apparatus 20 may include any device, such as asonicating or a vortexing apparatus (not shown), or the like to dispersethe bioactive component 12 in the hydrophilic composition, and theformation of the surfactant micelles 22 after addition of the surfactantcomposition 16. Nonetheless, while the first dispersing apparatus 20 mayinclude a sonicating or a vortexing apparatus, the sonicating or thevortexing apparatus is not critical when practicing the method of thepresent invention.

[0075] As used herein, a “surfactant micelle” may be characterized as aclose packed mono-molecular barrier of surfactant molecules at aninterface between the bioactive composition 12 and the surfactantcomposition 16, such that the barrier encapsulates the bioactivecomponent 12, the first aqueous composition 14 or the hydrophiliccomposition. It is also to be understood that the term “surfactantmicelle” encompasses partial or hemi-surfactant micelles that partiallyenclose the bioactive component 12, the first aqueous composition 14 orthe hydrophilic composition.

[0076] When the bioactive component 12 is a hydrophilic bioactivecomponent, the polar portion of the surfactant molecule associates witha surface of the hydrophilic bioactive component. When the bioactivecomponent 12 is a hydrophobic bioactive component, the hydrocarbonportion of the surfactant micelle associates with a surface of thehydrophobic bioactive component.

[0077] The formation of a surfactant micelle typically occurs at a welldefined concentration known as the critical micelle concentration. Asnoted, surfactant components having a CMC value of less than about 200micromolars are preferred when practicing the present invention.

[0078] After forming the dispersion of surfactant micelles 22, thedispersion of surfactant micelles 22 is transferred into the stabilizingapparatus 26 where a biocompatible polymer component 24 is added tostabilize the dispersion of surfactant micelles 22. Alternatively, thebiocompatible polymer component 24 may be added to the dispersion ofsurfactant micelles 22 in the first dispersing apparatus 20 whichobviates the need for the stabilizing apparatus 26.

[0079] The biocompatible polymer component 24 stabilizes the dispersionof surfactant micelles 22 to form stabilized surfactant micelles 28within the first aqueous composition 14. Therefore, a dispersion ofstabilized surfactant micelles 28 are present within the first aqueouscomposition 14 after addition of the biocompatible polymer component 24.

[0080] As used herein, the term “biocompatible” refers to a materialthat is capable of interacting with a biological system without causingcytotoxicity, undesired protein or nucleic acid modification oractivation of an immune response.

[0081] The biocompatible polymer component 24 may be introduced into thedispersion of surfactant micelles 22 as a liquid, vapor or in granularform. The form of the biocompatible polymer component 24 that isselected preferably permits the biocompatible polymer component 24 to(1) remain stable prior to addition into the dispersion of surfactantmicelles 22, (2) be homogeneously dispersed into the dispersion ofsurfactant micelles 22, (3) increase a viscosity of the first aqueouscomposition 14, (4) form a boundary layer at an interface of thesurfactant micelle 22 and the first aqueous composition 14, (5) beabsorbed onto a surface of the surfactant micelles 22, (6) be capable ofiontophoretic exchange, (7) be capable of being precipitated uponaddition of a solute, (8) be capable of enzymatic degradation, surfaceand/or bulk erosion, (9) not interfere with or mask the functionalactivity of the bioactive component 12, (10) prevent aggregation and/oragglomeration of the dispersion of surfactant micelles 22, and (11) becapable of obtaining a particular dissolution profile.

[0082] The biocompatible polymer component 24 may be included at anamount that is effective to coat and therefore stabilize the surfactantmicelle 22. Furthermore, those of ordinary skill in the art willrecognize that the amount of the biocompatible polymer component 24 usedto stabilize the surfactant micelles 22 may vary depending upon thebioactive component 12, the first aqueous composition 14, the surfactantcomposition 16, the temperature, pH, osmolarity, presence of anyoptional solutes or optional solvents, the surfactant micelle 22, anydesired characteristics of the stabilized surfactant micelle 28, thenanocapsules 36, or a desired dissolution profile.

[0083] While the concentration of the biocompatible polymer component 24is not critical to the present invention, the concentration of thebiocompatible polymer component 24 is preferably based upon thebioactive component and on the desired dissolution profile. When theconcentration of the biocompatible polymer component 24 is to high, theshell of the nanocapsule 36 may not dissolve. If the concentration ofthe biocompatible polymer component 24 is to low, the shell of thenanocapsule 36 may dissolve rapidly in a manner that promotescytotoxicity, for example. In addition, too low a concentration of thebiopolymer component 24 may not provide an effective degree ofmechanical force to stabilize the surfactant micelles 28.

[0084] Concentrations of the biocompatible polymer component 24 that areto high may also be less desirable because such higher concentrationsmay increase the viscosity of the first aqueous composition 14, andconsequently may cause difficulties in preparing, mixing and/ortransferring the stabilizer surfactant micelles 28. Concentrations ofthe biocompatible polymer component 24 that are to low may be lesspreferred, because lower concentrations may not provide the neededviscosity to stabilize the surfactant micelles, nor be capable ofeffectively coating the surfactant micelles 22 to prevent aggregation ofthe surfactant micelles 22 in the first aqueous composition 14.

[0085] The biocompatible polymer component 24 may be supplied asindividual biocompatible polymers or supplied in various preparedmixtures of two or more biocompatible polymers that are subsequentlycombined to form the biocompatible polymer component 18. Somenon-exhaustive examples of biocompatible polymers include polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate, cellulose propionate, cellulose acetate butyrate,cellulose acetage phthalate, carboxylethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexlmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene, polyvinylpryrrolidone, polyhyaluronic acids,casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexlmethacrylate), poly(isodecl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadeclacrylate) and combination of any of these.

[0086] Additionally, biocompatible polymers that have been modified forenzymatic degradation, or change upon application of light, ultrasonicenergy, radiation, a change in temperature, pH, osmolarity, solute orsolvent concentration may also be included as part of the biocompatiblepolymer component 24. Preferably, the biocompatible polymer component 24is a hydrophilic polymer that is capable of substantially coating, andpreferably continuously coating the surfactant micelle 22. Still morepreferably, the hydrophilic biocompatible polymer component 24 iscapable of ionotophoretic exchange.

[0087] Though descriptions of the present invention are primarily madein terms of a hydrophilic biocompatible polymer component 24, it is tobe understood that any other biocompatible polymer, such as hydrophobicbiocompatible polymers may be substituted in place of the hydrophilicbiocompatible polymer, in accordance with the present invention, whilestill realizing benefits of the present invention. Likewise, it is to beunderstood that any combination of any biocompatible polymer may beincluded in accordance with the present invention, while still realizingbenefits of the present invention.

[0088] In general, any conventional apparatus and technique that issuitable for permitting the biocompatible polymer component 24 tostabilize the surfactant micelles 22 may be used as the stabilizingapparatus 26 in accordance with the present invention. Furthermore, anyother device, such as high pressure homogenization or high ultrasoundsonication is preferably not included during stabilization.

[0089] After stabilizing the surfactant micelles 22, the stabilizedsurfactant micelles 28 may be transferred into a second aqueouscomposition 30 located in a second dispersing apparatus 32. Thestabilized surfactant micelles 28 may be transferred by mechanicallyforming droplets of the stabilized surfactant micelle 28 that aresubsequently introduced into the second aqueous composition 30.

[0090] The second aqueous composition 30 may include water only, or mayoptionally include a solute to precipitate the biocompatible polymercomponent 24 surrounding the stabilized surfactant micelle 28. Somenon-exhaustive examples of solutes that may be used to precipitate thebiocompatible polymer 24 include ionic species derived from elementslisted in the periodic table.

[0091] Preferably, the second aqueous composition 30 includes a solutein an amount that is effective to precipitate the biocompatible polymercomponent 24 and form the dispersed, and optionally atomizednanocapsules 36 of the present invention. As used herein, the term“precipitate” refers to a solidifying or a hardening of thebiocompatible polymer component 24 that surrounds the stabilizedsurfactant micelles 28. It is also to be understood that the term“precipitation” is also meant to encompass any crystallization of thebiocompatible polymer 24 that may occur when the biocompatible polymercomponent 24 is exposed to the solute.

[0092] Additionally, any other component that is capable of modulatingthe efficacy the nanocapsules 36 may be included as part of the secondaqueous composition to thereby modulate the functional activity of thenanocapsule 36. For example, the second aqueous composition may includeadditional coating excipients, such as a cell recognition component orvarious ionic species, such as Mn²⁺, Mg²⁺, Ca²⁺, Al³⁺, Be²⁺, Li³⁰ ,Ba²⁺, Gd³⁺, or any other ionic species that is capable of interactingwith the biocompatible polymer component 24.

[0093] The term “cell recognition component”, as used herein, refers toa molecule capable of recognizing a component on a surface of a targetedcell. Cell recognition components may include an antibody to a cellsurface antigen, a ligand for a cell surface receptor, such as cellsurface receptors involved in receptor-mediated endocytosis, peptidehormones, and the like.

[0094] It has been observed that when the stabilized surfactant micelles28 are allowed to incubate in the second aqueous composition 30 thatincludes the solute to precipitate the biocompatible polymer component24, the nanocapsules 36 undergo a reduction in size. Furthermore, theformation of a flocculated suspension of the nanocapsules 36 has alsobeen observed after incubating the stabilized surfactant micelles 28 inthe second aqueous composition.

[0095] As used herein, “a flocculated suspension” refers to theformation of a loose aggregation of discrete particles held together ina network-like structure either by physical absorption of bioactivecomponents, bridging during chemical interaction (precipitation), orwhen longer range van der Waals forces of attraction exceed shorterrange forces of repulsion. The flocculated suspension of nanocapsules 36may entrap varying amounts of the first aqueous composition 14 or thesecond aqueous composition 30 within the network-like structure.Additionally, the flocculated suspension of nanocapsules may be gentlytapped to disperse the nanocapsules 36.

[0096] The stabilized surfactant micelles 28 may be transferred into thesecond aqueous composition 30 via atomization through a nozzle (notshown) having a particular orifice size or through an aerosolizingapparatus (not shown). Atomizing or aerosolizing the stabilizedsurfactant micelles 28 typically includes the application of a shearforce that may be capable of further dispersing the stabilizedsurfactant micelles 28. Furthermore, the application of the shear forceduring transfer may also be effective to (1) reduce the size of thenanocapsules 36, or (2) break up any agglomerates or associationsbetween stabilized surfactant micelles 28 that may have formed in thestabilizing apparatus 26. Nozzle pressures of less than about 100 psi,for example, may be used to atomize the stabilized surfactant micelles28.

[0097] The diameter of the nanocapsules 36 may also be varied dependingupon the orifice size of the nozzle that may be used to transfer thestabilized surfactant micelles 28 into the second aqueous composition.Alternatively, the stabilized surfactant micelles 28 may be added to thesecond aqueous composition 30 containing the solute that precipitatesthe biocompatible polymer 24 to form a dispersion of nanocapsules 36 forpurposes of providing the dispersion for sub-cutaneous delivery of thenanocapsules, for example.

[0098] After precipitating and/or optionally incubating the nanocapsules36 in the second aqueous composition 30, the nanocapsules 36 may befiltered, centrifuged or dried to obtain separate and discretenanocapsules 36. The nanocapsules 36 may be frozen or reconstituted forlater use or may be delivered to a target cell or tissue by such routesof administration as oral, intravenous, subcutaneous, intraperitoneal,intrathecal, intramuscular, inhalational, topical, transdermal,suppository (rectal), pessary (vaginal), intra urethral, intraportal,intrahepatic, intra-arterial, intra-ocular, transtympanic, intraumoral,intrathecal, or any combination of any of these.

[0099] The nanocapsules 36 having a diameter of less than about 50 nmare advantageous in the delivery of bioactive components to target cellsfor several reasons. First, nanocapsules 36 having a diameter of lessthan about 50 nm enhances delivery of bioactive components by protectingthe bioactive components against degradation during transport to thetarget cell.

[0100] Second, nanocapsules 36 having a diameter of less than about 50nm promotes efficient cellular uptake. Efficient cellular uptake intothe target cell typically occurs when a particle has a diameter of lessthan about 50 nm, as opposed to when a particle has a diameter of morethan about 50 nm.

[0101] Third, it is believed that uptake of the nanocapsules 36 by thetarget cell occurs via transport systems, such as a non-endosomalpathway, that prevents lysosomal degradation of the nanocapsules 36.Indeed, it is believed that the nanocapsules 36 of the present inventionare efficiently exported into a cell via a caveolin-regulated pathwaythat circumvents most, if not all, endosomal-regulated pathways thattypically degrade nanocapsules 36.

[0102] Fourth, the nanocapsules 36 have a biocompatible polymer shellthat is separate from the bioactive component. In fact, the bioactivecomponent is not entangled in, embedded in, or adsorbed onto thebiocompatible polymer shell of the nanocapsules 36. When the bioactivecomponent is not entangled in, embedded in, or adsorbed onto thebiocompatible polymer shell, the cell that incorporate the nanocapsules36 avoid apoptosis or cell death.

[0103] Fifth, enclosing the bioactive component within a core surroundedby the biocompatible polymer shell when preparing the nanocapsules 36 inaccordance with the present method is advantageous in avoiding prematuredegradation of the nanocapsules 36, or a cytotoxic response during invivo transport of the nanocapsule. Enclosing the bioactive componentwithin the core results in a linear release rate of the bioactivecomponent without any zero burst phenomenon during release from thenanocapsules 36.

[0104] The linear release rate of the bioactive component from thenanocapsule without any zero burst phenomenon is also an advantageousfeature as the linear release rate allows rational design of coatingdissolution profiles to minimize cytotoxicity. As used herein, the term“dissolution profile” refers to a rate at which the biocompatiblepolymer shell is dissolved or degraded to release a bioactive agent froma core of a nanocapsule.

[0105] Another benefit of the nanocapsules 36 prepared by the method ofthe present invention is that little, if any, addition of an organicsolvent is required to form the nanocapsules 36. Eliminating the use ofmost, if not all, organic solvents from the method of the presentinvention is beneficial since organic solvents may damage the bioactivecomponent 12, destroy the target cells, or be toxic during preparationof the nanocapsule 36. The elimination of most, if not all, use oforganic solvents eliminates the need for complex solvent removaltechniques, such as solvent dilution, vacuum evaporation, or the like,and obviates any associated costs or complex process strategies duringpreparation of the nanocapsules 36.

[0106] The nanocapsules 36 of the present invention further permitsstable encapsulation of a bioactive component, and in particular,hydrophilic bioactive components, such as polynucleotides andpolypeptides. “Stable encapsulation”, as used herein, refers tomaintenance of the encapsulated bioactive component's structure. Fornucleic acids, the appearance of low molecular weight nucleic acidbreakdown products, which may be assayed for by electrophoresis, issubstantially eliminated. The nanocapsules 36 may also be used toencapsulate any bioactive component regardless of water solubility orcharge density.

APPLICATIONS

[0107] The nanocapsules 36 may be combined with additional polymericbinders, surfactants, fillers, and other excipients to incorporate thenanocapsules 36 into solid dosage forms such as granules, tablets,pellets, films or coatings for use in enhanced bioactive component 12delivery. In this way, design of the dissolution profile, control of theparticle size, and cellular uptake remains at the level of thenanocapsule. Such applications include, but are not limited to, creationof rapidly dissolving nanocapsule pellets for pulmonary delivery ornanocapsule films for device-mediated delivery.

[0108] In another application, the nanocapsules 36 may be designed forspecific cellular or tissue uptake by polymer selection and/or inclusionof cell-recognition components in the nanocapsule biocompatible polymershell or coating. Such coatings will have utility for specific orincreased delivery of the bioactive agent to the target cell. Suchapplications include, but are not limited to tumor-targeting ofchemotherapeutic agents or anti-sense DNA, antigen delivery toantigen-presenting cells, ocular delivery of ribozymes to retinal cells,transdermal delivery of protein antibodies, or transtympanic membranedelivery of peptide nucleic acids.

[0109] Property Determination and Characterization Techniques

[0110] Various analytical techniques are employed herein. An explanationof these techniques follows:

[0111]FIG. 1A: Samples were prepared on freshly cleaved mica asdispensed, dried in air and imaged using a Nanoscope II multimode AFM(Digital Instruments) with a J type scanner and ambient tapping modeholder. 125 μm long silicon cantilevers type IBMSC were from IBM andhave resonant frequencies of 250-450 kHz. All imaging was in tappingmode, images were 512×512 pixels and scanning frequency was 1 kHz.Height, amplitude and phase images were collected. Images were processedin DI software and analyzed in NIH Image SXM. A: Formula Q from 2-phasesystem, low HLB surfactant, B: Formula S from 2-phase system, high HLBsurfactant, C: Formula T from 1-phase system, high HLB surfactant, D:Formula V from 2-phase system, surfactant below CMC.

[0112]FIG. 1B: Nanocapsules were released into a solution of 10%isobutanol in Phosphate-buffered Saline (PBS), pH=7.2. Samples were runin duplicate. FIG. 1C: Nominal 300 ng samples of DNA were aliquoted froma master batch containing surfactant and processed through commercialminiprep columns. Eluate was recycled through Qiaquik columns andcollected either 3 times (4, 5) or twice (6,7) or recycled throughZymoclean columns and collected twice (8,9). Samples were alcoholprecipitated using a commercial coprecipitant, electrophoresed on 1.5%agarose gels modified with Synergel, stained with SybrGold dye,digitized on a Storm 860 and compared to unmodified but reprecipitatedsamples from the same master batch (10,11). Lanes 1-3: 100, 50 and 5 ngof ?_DNA.

[0113]FIG. 2: Endocytic activity was assessed by immunosignal levels ofclathrin (Chemicon). Potocytotic activity was assessed by immunosignalfor caveolin-1 as described in the literature (TransductionLaboratories). Lysosomal activity was detected by an monoclonal antibodyto Lamp-1 (Transduction Laboratories). Nanocapsule localization wasdetected by streptavidin-biotin immunocomplexes directed against sheepIgG (Jackson Laboratories). Nanocapsule coatings were spiked with ovineIgG to enable this detection strategy.

[0114]FIG. 3: immortalized Rt-1 fibroblast cultures at 70% confluencewere treated for 4 days with increasing amounts of nanocapsule formula Kand transiently treated (3 hours) with an optimized liposomal formula(dose, 500 ng) Results are expressed as a percentage of cellular actinintegrated intensity and compared to the liposomal formula. Expressionvector was code 448: pEF/myc-his/GFP (Invitrogen).

[0115]FIG. 4A: Radiated porcine biopsies were snapfrozen 7 days aftertreatment with saline or 6 μg of controlled release nanocapsules, thenhomogenized in RIPA. 100 μg lysate samples were electrophoresed onSDS-Page gradient gels, transferred to nitocellulose membranes anddetected for either §-galactosidase (121 Kd) or involucrin (˜100 kD)using chemiluminescence. Results were normalized to the post-transfergel stained with Coomassie due to interference at 100 kD from a geldefect. Involucrin, a component of the cornified membrane, manufacturedby suprabasal cells can be detected in radiated porcine skin and will beused for future normalization purposes. Lane A: N, topical, biopsy oc-2;B: N, topical, biopsy oc-3; C: 0, topical, biopsy 1-1; D: PBS only,biopsy 1-5; E: N, subcutaneous injection, biopsy 1-6.

[0116]FIG. 4B: The β-galactosidase reporter protein was detected by amonoclonal antibody directed at an incorporated fusion protein tag. A:N, topical, biopsy oc-1, detection with anti-Xpress^(a); B: Matchingview to A with detection for anti-von Willenbrand factor (Sigma);C:untreated biopsy, detection with anti-Xpress^(a)(Invitrogen).

[0117]FIG. 5: Nanocapsules were incorporated into an aqueous suturecoating and sutures were applied to pigskin biopsies in organ culture.Nanocapsules were detected with Cy3 conjugated-streptavidin-biotincomplexes to incorporated ovine IgG and GFP transgene expression wasdetected by rabbit polyclonal antibodies to GFP (Abcom) in combinationwith Fitc-conjugated polyclonal antibodies to rabbit IgG and Alexa488-conjugated polyclonal antibodies to Fitc (Molecular Probes).Controls omitting primary antibodies were included forsignal-to-background level estimation.

[0118]FIG. 6A: Nanocapsules were detected as previously described andGFP transgene expression was detected by rabbit polyclonal antibodies toGFP in combination with Cy3-conjugated antibodies to rabbit IgG (JacksonLaboratories).

[0119]FIG. 6B: GFP expression was detected as described in FIG. 5 andcell nuclei were counterstained with 10 μg/ml bisbenzamide.

[0120]FIG. 6C: Carcinoma cells and HDF's were seeded overnight into 96well plates at 2000 and 6000 cells per well respectively. Cisplatinpreparations were added to wells for 18 hours as noted on the graph thanwashed out. After 72 hours cell viability of assessed by a commercialMTT assay (WST assay, Boehringer Mannheim). Wells were executed induplicate.

[0121]FIG. 7: Colocalization with lysosomes was detected using amonoclonal antibody to Lamp-1 (Transduction Laboratories). AFM imagesare included of O-methyl RNA formulated by nanoencapsulation orcomplexation with 27 KD polyethyleneimine.

EXAMPLES

[0122] The present invention is more particularly described in thefollowing Examples which are intended as illustrations only sincenumerous modifications and variations within the scope of the presentinvention will be apparent to those skilled in the art.

[0123] Reagents:

[0124] A. Nucleic acid condensing agents

[0125] Poly(ethylenimine) (PEI) at 27 KiloDalton (kD). PEI was used atoptimized conditions (90% charge neutralization)

[0126] Polylysine (PLL) at 70-150,000 molecular weight. PLL condensingmaterials were conjugated with nuclear signal localization peptides,either SV-40 T antigen orcys-gly-tyr-gly-pro-lys-lys-lys-arg-lys-val-gly-gly using carboxiimidechemistry available from Pierce Chemical (Rockford, Ill.).

[0127] Preparations of nuclear matrix proteins (NMP). NMP were collectedfrom a rat fibroblast cell line, and a human keratinocyte cell lineusing a procedure described in Gerner et al. J. Cell. Biochem. 71(1998):363-374 which is incorporated herein by reference. Proteinpreparations were conjugated with nuclear signal localization peptidesas described.

[0128] B. Surfactants

[0129] 2, 4, 7, 9 - tetramethyl-5-decyn-4, 7 - diol (TM-diol): HLB=4-5,CMC is not determined

[0130] Poly(oxy-1, 2-ethanediol), a-(4-nonylphenol)-w-hydroxy, TergitolNP-40 (NP40): HLB=17.8, CMC 180 μM,

[0131] Polyoxyethylene 20 sorbitan monooleate (Tween 80): HLB=10, CMC920 μM, Cetyl Alcohol: HLB=4,CMC is not determined.

[0132] C. Polymers

[0133] Hyaluronan, bacterially-derived, 1 million kiloDalton (MM kD) andconjugated with nuclear localization signal peptides as described inU.S. Pat. No. 5,846,561, which is incorporated herein by reference.

[0134] Hyaluronan, derived from human umbilical cord, about 4MMKD andnot conjugated.

[0135] Povidone (polyvinylpyrolidone, PVP) 10,000 kD MW and notbioconjugated Povidone (polyvinylpyrolidone, PVP) 40,000 Kd MW and notbioconjugated Povidone (polyvinylpyrolidone, PVP) 360,000 kD MW and notbioconjugated Tenascin, 220 kD and not bioconjugated.

[0136] D. Expression Vectors

[0137] 334: pcDNA/His/LacZ, produces galactosidase, incorporates CMVpromoter, based on pcDNA 3.1. (Invitrogen), 8.6 kB

[0138] 425: pEGFP-c/farn, enhanced GFP (green fluorescent protein)expression vector modified with a farnasyl moiety to improve microscopy,CMV promoter, 4.6 kB

[0139] 423: pEGFP-c3/p57(Kpn/Sma) Clontech enhanced GFP (greenfluorescent protein) expression vector modified with a nuclearlocalization tag from a cyclin dependent kinase to improve microscopy,4.6 kB

[0140] E. Cells

[0141] CCRL 1764: Immortalized rat neonatal fibroblast cell line

[0142] HaCaT: immortalized human keratinocyte cell line

[0143] Ca9: human tumor cells derived from a squamous cell carcinoma oftongue origin.

Example 1A—Effect of changing dispersion conditions on hydrophillicnanocapsules.

[0144] The importance of appropriate dispersion conditions wasinvestigated in the following series of formulations. Formulae wereproduced by i) predispersing 25 μg of DNA (425) on ice using a bathsonicator, ii) condensing DNA in a small amount of water by vortexingthen incubating on ice for 20 minutes, iii) adding surfactant then oilfollowed by 30 seconds of probe sonication at 10 Watts, iv) diffusiondilution to 3 milliliters (mL) by first adding saline then 1 MM kDhyaluronan polymer (1%) as a protective colloid, v) mechanicallyshearing emulsion into droplets by pumping through a 250 micrometer (μm)orifice into 22 mL of PBS, 10 milliMolar (mM) Ca²⁺, 200 mM Li⁺, vi)incubating overnight end over end and vii) centrifuging to recovernanoparticles for resuspension and filter sterilization. Thecondenser-to-DNA weight ratio was determined by dye exclusion at 90%charge neutralization. TM-diols were used in this experiment torepresent water-immiscible surfactants, while Tergitol NP40 and Tween 80were used to represent water-soluble and even more water-solubleemulsifiers/dispersing aids.

[0145] Dispersion conditions were systematically varied to discouragemicelle formation in aqueous media by i) choosing water-solublesurfactants (Formulae S,T,U, and V), ii) removing the dispersed phase(Formula T) and iii) decreasing surfactant loading below that requiredfor micelle formation. Formula U featured use of a water-miscible oil(silicone oil).

[0146] Formulas were characterized physically and tested forfunctionality in in vitro gene transfer. Quantitative results aresummarized in Table 1A: TABLE 1A Effect of changing dispersionconditions on hydrophillic nanocapsules. Formula Q R S Experimentalsurf > CMC surf > CMC surf > CMC Modification: Critical Micelle ˜0 ˜0360 ppm Concentration (CMC) Pre-aerosol surfactant 500 ppm 500 ppm 600ppm Concentration (3 ml basis) HLB number 4-5 4 17.8 Phases Water/Water/ Water/ misc. oil misc. oil misc. oil Formula Characteristics:Nucleic Acid 86 ± 8   67 ± 1.4 50.3 ± 12   Incorporation (%) Low MW DNA15.00 76 93.00 Appearance (% above bkground, Post nanocapsule digest byelectrophoresis) Supercoil retention 87% 65% 66% (post 100 hrs release)(area %, initial distribution = 76% supercoiled) Particle Size 42 ± 2 45 ± 3  73 ± 4  (mean ± SE) Secondary Structure(s) 25% 30% 70%Flocculation Status 100-200 nm 500 nm 300 nm stringy flocs stringy flocsspheroid aggregates Comments: Performance: Transduced GFP 420 340 0Protein Generation (pixel units, % of control liposome formula, 100 μgtotal protein, Day 11)

[0147] TABLE 1A Effect of changing dispersion conditions on hydrophillicnanocapsules. Formula T U W V Experimental surf > CMC surf > CMC surf >CMC surf < CMC Modification: Critical Micelle 360 ppm 360 ppm 1200 ppm360 ppm Concentration (CMC) Pre-aerosol 600 ppm 600 ppm 4000 ppm  90 ppmsurfactant Concentration (3 ml basis) HLB number 17.8 17.8 10 17.8Phases Water only Water/immisc. oil Water/misc. oil Water/misc. oilFormula Characteristics: Nucleic Acid  39 ± 1.7 32.8 ± 6     37 ± 1.4157.6 ± 16   Incorporation (%) Low MW DNA 53.00 66 28 41.00 Appearance (%above bkground, Post nanocapsule digest by electrophoresis) Supercoilretention 59% 43% 65% 80% (post 100 hrs release) (area %, initialdistribution = 76% supercoiled) Particle Size (mean ± SE) 226 ± 11  291± 25  150 ± 7  199 ± 11  S < 10% S < 10% S > 40% S > 80% 400 nmSecondary yeast-like aggregates Structure(s) aggregates FlocculationStatus ppt. during ppt. during ppt. during Comments: aerosolizationaerosolization aerosolization Performance: 0 0 0 0 Transduced GFPProtein Generation (pixel units, % of control liposome formula, 100 μgtotal protein, Day 11)

[0148] Nanocapsule sizing was determined by tapping mode AFM and imagesare illustrated in FIG. 1A. The data indicate average nanocapsule sizesless than 50 nm are achievable only with multi-phase systems incombination with low water solubility surfactants (Table 1A: FormulaeQ,R vs. S,T,U,V, and W). Furthermore, only nanocapsules of less than 50nm resulted in detectable transgene production in CRL-1764 ratfibroblast cells (Table 1A). Effective dispersion also corresponded withdecreased aggregation and enhanced DNA stability (as indicated bydecreased electrophoretic breakdown products). The starting DNA waspartially relaxed (76% supercoiled by electrophoresis). Using this valueas a basis, supercoil retention in DNA still encapsulated following 100hrs of release testing, was excellent in multi-phase systems.

[0149] Release profiles for hydrophillic dispersed atomized nanocapsuleswere linear, showed no zero burst and resulted in about 60% releaseafter 72 hours (See FIG. 1B). Formula W, manufactured with the mostwater-soluble surfactant in the series (Tween 80) failed to completelyrelease loaded DNA. FIG. 1C illustrates that small amounts of DNA (inthis case 300 nanograms of DNA) can be recovered accurately in aprocedure comprising butanol extraction of 10% butanol/saline releasingfluid followed by isolation on a miniprep column and measurement ofabsorbance at 260 nm excitation. Results obtained from UV spectroscopyare confirmed by electrophoresis of recovered DNA following alcoholcoprecipitation with a commercial coprecipitant aid. Experiment 1Ademonstrates the importance of a multi-phase system in creating coatedparticles from the micellar solution, defines surfactant requirementsand validates method for measuring in vitro release profiles.

Example 1B—Effect of process parameters on particle functionality

[0150] To investigate the effect of modulating process parameters onnanocapsule functionality for DNA delivery, a series of formulas(designed to release in 3 days) were prepared and measured transductionefficiency of these formulas for delivering a nuclear Green FluorescentProtein (GFP) reporter transgene in rat fibroblast cultures 5 dayslater. Charge neutralization of the DNA molecule, the surfactant/oilsystem, total surfactant phase volume, the inclusion of probesonication, the absolute requirement for atomization and receiving bathosmolality were modulated. Results for this experiment are summarized inthe Table 1B: TABLE 1B Effective of process parameters on particlefunctionality Oil Receiving Nano charge Phase Atomize bath capsuleFormula neutralization by Biocompatible Volume Emulsify by DiameterOsmolality Design Name condensor Surfactant Oil (%, 4.5 ml basis)sonication (μm) (mOs) 1 q.co.2 + Cetyl Castor 4 + 250 220 OH oil/Etoh 2q.co — Cetyl Castor 4 + 250 220 OH oil/Etoh 3 o.35 + TM-diol DMSO 4 +1.4 220 4 ea0.2 + TM-diol DMSO 4 — — 220 5 ea0.1 — TM-diol DMSO 4 — —220 6 ed0.2 + TM-diol DMSO 0.05 — 250 220 7 ed0a.12.di + TM-diol DMSO0.05 — 250 0 Transduction Nanocapsule Encapsulation Efficiency, (5Nanocapsule Formula diameter (nm)* yield days, rat Design name n = 20(%, mean ± SE) fibroblasts) 1 q.co.2 20 ± 3, rods 48.6 ± 11 87 ± 7% 2q.co 12 ± 0.7, irregular 48.6 ± 2 71 ± 28% 3 o.35 17 ± 1.2, spheres 82.3± 7 (4) 86 ± 2% 4 ea0.2 24 ± 2, s/r   32 ± 10 72 ± 2% 5 ea0.1 36 ± 3,irregular   57 ± 2 85 ± 1% 6 ed0.2 39 ± 3, r/e   39 ± 5  96% 7 ed0a12.di39 ± 3, ellipse   69 ± 2 100%

[0151] Aqueous dispersion of DNA condensates with poorly solublesurfactants in the inventive method produced average nanocapsulediameters under 50 nm. A 30 number of successful operating regimes werefeasible with varying effects on encapsulation yield. In a cetylalcohol/castor oil system, under condensation resulted in an averageparticle diameter increase from 20 to 12 nm (Table 1B: F1vs. F2). Thesame decrease in condenser weight ratio induced a particle size increasefrom 24 to 36 nm, while still maintaining nanocapsule functionality fortrangene delivery, when using a TM-diol/DMSO surfactant system forinitial micelle formation (Table 1B: F4 vs. F5). This finding teachessurfactant selection impacts final average nanocapsule diameters.

[0152] Moderate energy input was removed (dropped probe sonication,atomization but kept bath sonication) during nanocapsule formation andresulted in functional particles with decreased yield (Table 1B: F3 vs.F4). This finding indicates that optimal nanocapsule production is notdependent on any spontaneous micro-emulsification process. Cosolventphase volume was reduced from 4 weight percent to 500 ppm without anynegative effect on particle functionality (Table 1B: F4 vs. F6). Thisfinding indicates that essentially solvent-free nanocapsules can be madeby the inventive method. Finally, salt was removed from the atomizationreceiving bath without any negative effects on nanocapsule functionality(Table 1B: F6 vs. F7).

Example 2—Effect of nanocapsule sizing on a nanocapsule uptake in humankeratinocytes

[0153] The effect of nanocapsule sizing on intracellular trafficking inimmortalized HacaT human keratinocyte cultures (HacaT's) wasinvestigated in this example. In this series of formulae, thee micellardispersion were sheared by syringes of different orifice diameter. Thecoating weight was also lowered from 1:1 DNA:Polymer (w/w) to 1:40 toshorten the dissolution profile from 5 to 3 days. In these experiments,nanocapsule formulae were compared to standard polyplexes of DNA andPEI, and lipoplexed plasmid DNA. Table 2 summarizes the experimentaldesign and results: TABLE 2 Effect of particle size on nanocapsulefunctionality for gene transfer 4 hr. 4 hr. 10 hr. Transduction ParticleSize colocalization colocalization colocalization Efficiency. Formula(mean, nm; with with with (5 days, human Name morphology) caveolin-l*cathrin lysosomes keratinocytes) o.22 (64) 47 ± 3, rods 0 ++ + 16 ± 13o.27 (57) 21 ± 2, rods + + ND 81 ± 8 o.35 (85) 17 ± 1.2, spheres +++ 0 078 ± 9 pei- 67 ± 4, 0 +++ +++ 40 ± 15 pDNA spheres, irreg Lipoplex 48 ±2 + + +++ 41 ± 27 pDNA 200 nm aggregates

[0154] It was observed that compared to the unstimulated state,nanocapsules increased cellular pinocytotic activity compared tostandard formulations, and smaller nanocapsules shifted pinocytoticactivity to caveolae from clathrin-coated pits (Table 2: Formula O vs.pei-dna and lipoplex pDNA). It was further observed that nanocapsulesavoided lysosome co-localization at 10 hours post-addition with smallernanocapsules being particularly effective (see Table 2: Formula vs.pei-dna and lipoplex pDNA). These results are illustrated further inFIG. 2. This improvement is further emphasized by comparison withpublished uptake studies for HacaT keratinocytes. Compared to primarykeratinocytes, uptake of naked DNA oligonucleotides (20 μm) were verypoor in HacaT's and showed accumulation of oligonucleotides in punctatevesicles consistent lysosomes at 2 hours. Using hydrophillic dispersedatomized nanocapsules of the inventive method, complete avoidance oflysosomes at 10 hours post-addition was demonstrated. These resultsindicate that products of the inventive process will have increased andprolonged effectiveness.

Example 3—Effect of nanoparticle delivery on DNA and reagent-inducedcytoxicity

[0155] To test whether soluble exogenous DNA released from liposomes ordendrimers induces apoptosis, Rt-1's were treated with loaded/unloadedliposome complexes, dendrimer complexes, nanoparticles and 1 μg/mletoposide, a DNA intercalating agent as a positive control. Cultureswere treated with standard formulas for 3 hours then assayed for geneproduct expression 30 hours later. Cultures were treated withnanocapsules for 4 days to ensure full DNA release during theexperiment. Controls included as a positive control for apoptotic celldeath, 1 μg/ml etoposide, a DNA intercalating agent was applied tocultures overnight before experiment termination. Other controlsincluded standard PEI-DNA complexes, empty nanocapsules and nanocapsulescontaining empty vector plasmid DNA. Hydrophillic nanoparticles wereproduced for this experiment as described earlier using a 35-gagesyringe.

[0156] One of the later steps in apoptosis is DNA fragmentation mediatedby activation of endonucleases during the apoptic program. Therefore,DNA fragmentation was assayed by end-labeling of fragments using anexogenous enzyme and a substituted nucleotide (TUNEL: tdt-mediateduridine nucleotide and labeling. Results are expressed as aFragmentation Index, or the percent of cells in the total cultureexhibiting BRDU end-labeled DNA. Cultures were run in duplicate. Theexperimental design and results are summarized in Table 3: TABLE 3Effect of nanocapsule coating weight on nonspecific reagent and plasmidDNA- associated cytoxicity. Formula κ.35 ζ ο(Omicron) b.35 ParticleDesign: DNA Condensing Denatured h. 100 Kd MW 27 kD 27 kD Agentkeratinocyte Polylsine PEI PEI nuclear protein Coating Ratio 0.1 0.250.25 0.01 (DNA/polymer) Performance: dose: (30 hrs for Std. 4.6 4.1 4 5Formulas, 100 hrs for nanocapsules) Cytotoxicity: ND  0.26 ± 0.15   2 ±0.7 1.9 ± 0.6 (Fragmentation Index, %) cytotoxicity controls: (1 μgetoposide (8 hr): 25 ± 10%) (Pei- DNA polyplexes (100 hr): 24 ± 7%)(Empty vector nanocapsules: 1.25 ± 1.25%) (Empty vector nanocapsules:0.9 ± 0.7%) Transduction 31 ± 2 ND 85 ± 7  32 ± 3  Efficiency: (% cells)120 hrs, dose as listed) Formula Characteristics: Nucleic: Acid  55 ± 1027 ± 7 54 ± 5  65 ± 4  Incorporation: (%) Cumulative Release: 70 75 ± 883 ± 12 ND (%, 48 hr) Particle Size (mean ± SE, 26 ± 2 22 ± 2 20 ± 1  35± 2  nm) Agglomerates (as few 50% 80 ± 6 200 nm 200 nm dispensed)Formula Y.35 Lipoplex GP Lipoplex L+ Polyplex Particle Design: DNACondensing 27 kD cationic cationic dendrimer Agent PEI lipid lipidCoating Ratio 0.0025 (DNA/polymer) Performance: dose: (30 hrs for 5  1μg 500 ng 2 μg Std. Formulas, 100 hrs 500 ng 250 ng 1 μg for  0 ng  0 ng0 μg nanocapsules) Cytotoxicity:  9 ± 8 27 ± 8 9.3 ± 0.2 6.63 ± 1.4 (Fragmentation Index, %) cytotoxicity  6 ± 3 12.8 ± 1.5  5.7 ± 1.8controls:   4 ± 2.5 7.8 ± 0.1 3.1 ± 0.3 (1 μg etoposide (8 hr): 25 ±10%) (Pei-DNA polyplexes (100 hr): 24 ± 7%) (Empty vector nanocapsules:1.25 ± 1.25%) (Empty vector nanocapsules: 0.9 ± 0.7%) Transduction 24 ±4 17 ± 2 dead dead Efficiency: (% cells) 120 hrs, dose as listed)Formula Characteristics: Nucleic: Acid  667 ± 0.2 ND ND NDIncorporation: (%) Cumulative ND ND ND ND Release: (%, 48 hr) ParticleSize 57 ± 5 48 ± 2 ND 22.4 ± 2   (mean ± SE, nm) Agglomerates (as g.t.50% 300 nm 25% 300 nm dispensed) 300 nm hard-fused

[0157] TABLE 3B Dose response of nanoencapsulated pDNA GFP/ActinProduction Formula Dose (100 hr.) (density ratio, %) K.35   9 μg 94.8K.35 4.5 μg 83.5 K.35 1.5 μg 83.3 Lipoplex GP o.5 μg 94.9

[0158] It was observed that use of controlled-release nanocapsulesreduced the fraction of apoptotic cells in fibroblast cultures 3 to 100fold. Conventional reagents without DNA showed a 4-fold increase in FIover empty nanoparticles, but increased another 50-100% withoutadditional reagents in the presence of additional DNA. Decreasing thecoating weight from 1:40 to 1:400 resulted in an increase in averagenanocapsule diameter from 20 to 57 nm and the appearance of regions ofapoptotic induction in cultures (Table 3: F omicron vs. F upsilon 35).Decreasing the coating weight from 1:40 to an intermediate 1:100 reducedtransduction efficiency without increasing particle size and theappearance of cytotoxicity. These findings indicate that nanocapsuledesign plays a role in maintaining nanocapsule integrity and that sizeeffects and dissolution profiles can contribute to observed cytotoxicityand functionality. We concluded that application of nanocapsuleformulations increased dosing to useful efficiency levels withoutinduction of an apoptotic program.

[0159] Table 3B exemplifies this improvement with a dose response ofFormula K.35 measured in fibroblast lysates. GFP production was measuredin fibroblast lysates after 4 days of treatment with increasing doses ofnanocapsules. A 9.5 μg dose of nanocapsules equaled the production of aliposomal formulation without any evidence of cytotoxicity.

Example 4—Nanocapsule delivery of macromolecules to porcine tissueacross keratinized barrier epithelia bv transdermal and subcutaneousmeans

[0160] The utility of nanocapsules for nonviral nucleic acid delivery totissue in a pig biopsy organ culture system was investigated. 6 and 8 mmcircular biopsies were collected under sterile conditions from sedatedresearch animals and cultured on meshes in partial contact with mediacontaining 20% Fetal Calf Serum. Biopsies were either injected with 90μl (6.3 μg) or treated topically with 3×30 μl aliquots. Biopsies weresnapfrozen 7 days later and sectioned/homogenized for β-galactosidaseproduction measurement. Formulation information and results from thisexperiment are summarized in Table 4: TABLE 4: Table 4: Functionality ofdispersed atomized nanocapsules for macromolecule delivery acrosskeratinized barrier membranes. Formula N O Exp. Modification coating wt.is 2.5x coating (from Formula Q) Polymer MW is 1x wt. is 2.5x Polymer MWis 4x Formula Characteristics: Nucleic Acid Incorporation (%) 70.0070.50 Cumulative Release 83 83. ± 1.5 (%, 169 hr 2.5 μg sample Low MWDNA in postdigested 0 0 Electrophoresis Samples Supercoil retention 100%100% (237 hr release, initial = 69.7% sc/releaxed) Particle Size (mean,SE, major 18.2 ± 0.2 nm ND species) Particle Description sphericalSecondary structure: 20% 100 nm flocs Performance: Transduced ProteinProduction 312 ± 74 (topical) (pixel units, 142 (s.c.) % of neg control,100 μg total protein, normalized by protein) Reporter (gene Product 100%100% Distribution (6.3 μg dose, 6 mm (N), 8 mm (O) porcine biopsy, 1 wk)keratinocytes (% cells), n = 2 73 ± 20 (pap) 13.8 ± 0.5 (pap) fields/200cells, neg cntrl: 6% endothelial cells, (% vwf-+area) papillary and/orreticular, 32 ± 15 (ret) 8 ± 2 (ret) n = 2-4 fields, neg cntrl: 1.07 ±0.72 dermis (% area); 2.74 ± 0.96* 1.77 ± 0.49* negative cntrl: 0.24 ±0.03, n = 4/20x fields

[0161] Western blotting of radiated tissue lysates showed a 3-foldincrease in β-galactosidase in duplicate biopsies treated topically withFormula N over an identically cultured 6mm biopsy treated with saline.Only a 2-fold increase was measured in a 8 mm biopsy treated topicallywith formula 0 nanoparticles (see FIG. 4B). Formula O was produced witha higher molecular weight analog of the N polymer suggesting adifference in particle morphology, a dose effect or differing in siturelease profiles between the two formulations related to thisdifference. To identify initial cell type-specific differences innanocapsule delivery effectiveness, tissue sections were analyzed forβ-galactosidase expression in double-label experiments using antibodiesto cell-specific epitopes (see FIG. 4B). Digital image analysis of thesesections indicated that radiated keratinocytes and endothelial cells arereadily transduced in organ culture 7 days after treatment with a 10 dayreleasing formula. Specific quantitation of fibroblastic cells was notpossible without inclusion of a cell-specific marker, however, an11-fold increase in area of expression was measured in N biopsy dermis(see FIG. 4B). Interestingly, for both the formulae N and Otopically-treated biopsies examined, the area percentage of bloodvessels transduced decreased about 50% in nearby fields between 100 μmand 300 μm of depth (Table 4: papillary vs. reticular endothelialcells). These data suggest that nanocapsules are penetrating theepidermis to enter the dermis.

Example 5—Incorporation of inventive nanocapsules into a solid dosageform for additional utility in physical targeting

[0162] Nanocapsules containing a nuclear GFP transgene or empty vectorwere incorporated into a suture coating by vortexing the followingcomponents: i) 50 μg of nanocapsules containing plasmid DNA, ii) 200 μgof bovine mucin, and iii) 75 μg of sucrose (60% w/w) in a 1000 μlvolume. Sutures were aseptically coated by drawing sutures 5× throughpunctured microcentrifuge tubes. Coating functionality for gene transferwas tested by applying sutures in cultured porcine skin biopsies.Biopsies were cultured on a mesh such that the biopsy bottom was incontact with cell culture media. Biopsies were treated for 7 days, thensnap-frozen and sectioned for immunofluorescence microscopy to assessnanocapsule penetration and transgene delivery.

[0163] Nanocapsule penetration was detected by streptavidin-biotinimmunocomplexes directed at sheep IgG. Nanocapsule coatings are spikedwith ovine IgG to enable this detection strategy. FIG. 5A showsdistribution of sheep IgG signal throughout porcine dermal tissue withaccumulation on capillaries. In FIG. 5A′, primary antibody is omittedduring slide processing to determine level of background fluorescence. Asuture is visible in this view. Sutures were identifiable as smoothobjects without positive nuclear counterstain. GFP expression wasconfirmed using a polyclonal GFP antibody to obviate the effect ofnonspecific tissue green fluorescence. FIG. 5B shows GFP expressionthroughout the suture-treated dermis using a GFP polyclonal antibody. Asuture was visible 750 microns away. FIG. 5C shows the lack of GFPexpression in a biopsy treated with empty vector coating. This exampledemonstrates the usefulness of nanocapsules for use in physicallytargeted macromolecule delivery.

Example 6—Utility of nanocapsules for local targeting by design ofnanocapsule coating

[0164] Fibroblast targeting

[0165] GFP nanocapsules were prepared by dispersion atomization asdescribed in Example 1. Polyvinylpyrylodone (PVP, MW 10,000) was used asthe coating basis. A coating weight ratio of 1:40 was used androd-shaped nanocapsules of 23±2 nm were produced. 1 μg of PVPnanocapsules were applied to both human dermal fibroblasts (HDF) andHacaT keratinocyte cultures for 4 hours then fixed for detection fornanocapsule uptake by streptavidin-bioting immunocomplexes to sheep IgG.Nanocapsule coatings are spiked with ovine IgG to enable this detectionstrategy. FIG. 6 illustrates positive nuclear localization of PVPnanocapsules in HDF's and negative colocalization of PVP nanocapsules inkeratinocytes (FIG. 6: 6 a vs. 6 b). Views of untreated cultures areincluded for comparison (6 a′, 6 b′). Cultures were also treated with 5μg of PVP nanocapsules for 5 days then tested for GFP transgeneproduction. Consistent with uptake studies results, only the fibroblastcultures showed production of GFP transgene (FIG. 6: 6 a″ vs. 6 b″).

[0166] Tumor-targeting

[0167] GFP nanocapsules were prepared by dispersion atomization asdescribed in example 1. Tenascin (TN, MW 200,000) was used as thecoating basis. A coating weight ratio of 1:20 was used and sphericalnanocapsules of 19±0.9 nm were produced. 500 ng of TN nanocapsules wereapplied topically in successive small aliquots to pig biopsiesmaintained in organ culture. Biopsies were rinsed in media after 3minutes of topical application and culture media was changed to precludeany delivery other than topical.

[0168] To simulate tumor nests of epithelial-derived origin, biopsieshad been seeded 12 hours previously with 50,000 human squamous carcinomacells. 7 days later biopsies were snapfrozen and sectioned forimmunological detection of GFP production. In FIG. 6B, view “a” showsintense GFP fluorescence in the tumor center, view “b” confirms this GFPexpression with polyclonal antibodies to GFP, view “c” shows cellpositioning in the section using a counterstain for cell nuclei and view“d” shows the level of background fluorescence by omission of GFPantibodies. Tumor origin was confirmed by positive detection withantibody to keratin 10/1, an epithelial marker. Comparison of view “b”and view “c” indicates that GFP expression is limited to cells withinthe tumor. As already demonstrated in example 5, expression throughout atissue is also feasible and can modulated by coating design. Thisexample demonstrates that nanocapsule delivery can be productivelytargeted.

[0169] Cell-specific delivery for enhanced drug therapeutic window

[0170] Nanocapsules were prepared as described in Example 1 toencapsulate cisplatin, a hydrophobic molecule and a common cancerchemotherapeutic with serious side effects. A coating weight ratio of1:100 was used and irregular nanocapsules of 29±3 nm were produced.Targeting efficacy was demonstrated by changes in the dose response forcell growth inhibition in fibroblast vs. squamous cell carcinomacultures. Cells were seeded overnight into 96 well plates, treated for18 hours with increasing amounts of encapsulated or unencapsulated drug,then assessed for cell growth inhibition using an MTT assay 48 hourslater for total growth time of 72 hours. Results are illustrated in FIG.6C. The data shows that tenascin nanocapsules protected nontarget cellsfrom cell death (zero death) at drug levels that killed usingunencapsulated drug (FIG. 6Aa: open vs. closed circles). In carcinomacultures, TN nanocapsules productively decreased the inhibitionconcentration (IC50) an estimated 300% from 525 to 160 μg/ml. Example 6demonstrates the usefulness of nanocapsules for use in coating-targetedmacromolecule delivery.

Example 7—Utility of nanoencapsulation for improved cellular uptake ofother species used as pharmaceutical, nutraceutical, research orcosmetic agents

[0171] Nanocapsules containing either 500 kD Fitc-labelled dextran, 20mer Fitc-labelled mer O-methylated RNA oligonucleotide and 16 merphosphodiester DNA oligonucleotide were prepared as described inExample 1. A 1:40 coating weight ratio was used and 1 MM kD hyaluronanwas used a coating basis. PEI was used to condense the phosphodiesterDNA oligonucleotide, but no PEI was included in the dextran or RNAoligonucleotide formulas. Nanocapsule functionality for drug deliverywas tested by evaluating changes in relative pinocytotic activity andcellular uptake in 35 mm cultures of human dermal fibroblast.Nanocapsule formulas were compared to naked species or speciesformulated as complexes. Quantitative results are summarized in Table 7.TABLE 6 Nanoencapsulation improves cellular uptake of other species usedas pharmaceutical, nutraceutical, research or cosmetic agents. At 18hours post-addition, lysosomes are only evident in conventionallyformulated species. 4.5 hours post-addition 18 hours post-additionIncrease in cellular Bioactive uptake activity, Nuclear component (%cells above Uptake Colocalization with Detection Particle size baseline,Efficiency lysosomes, persistence Bioactive (mean, SE, nm,caveolin-l/clathrin) (% cells, (% cells, human (% cell, human ComponentFormulation morphology) dose fibroblast) fibroblasts) fibroblast) 500 kdnanocapsule  22 ± 2, s/r 89/20  25 μg* 95 ± 2  2 ± 2  5 μg 88 ± 11flic-dextran naked, Fitclabelled — 75/18 100 μg  10 100 ± 10 100 μg 61 ±20  20 mer o- nanocapsule  13 ± 0.7, r 78/90  2 μg 74 ± 5  0 ± 0  5 μg80 ± 6 methylated naked, Fitclabelled — —/73  5 μg 14 ± 7 — — RNA oligoPEI/Fitclabelled 236 ± 26, r —/— — — 100 ± 0  5 μg 94 ± 10  16 mer POnanocapsule  17 ± 1, r 70/94  1 μg 34 ± 25  0 ± 0  5 μg 91 ± 8 DNA oligoPEI/Fitclabelled  67 ± 4, s/r 72%  2 μg 95 ± 2  80 ± 7  5 μg 66lysosomes Nominal n 20 particles 70 cells 140 cells 50 cells 50 cells

[0172] Table 7 shows that average diameters for all nanocapsules werebelow 50 nm by AFM. PEI complexes of DNA oligonucleotides were measuredat 67 nm and PEI complexes of uncharged RNA O-methyl oligonucleotideswere measured at 236 nm. As discussed in Example 2 using keratinocytecultures and plasmid DNA, nanocapsules stimulate pinocytotic activity asindicated by increased signal levels of clathrin and caveolin-1. In the500 kD dextran case, pinocytotic activity shifts productively towardscaveolae with nanoencapsulation (Table 7, 500 kD Dextran). At 4.5 hourspost-addition, nuclear uptake is enhanced for encapsulated dextran andRNA relative to naked species. For the nanocapsules of DNAoligonucleotides, cellular uptake is decreased relative to complexedoligonucelotide, however, a majority of that DNA oligonucleotides isalready nonproductively sequestered in lysosomes by that 4.5 hours(Table 7). At 18 hours post-addition, nanocapsules species showcontinued exclusion from lysosomes, while naked species show high levelsof sequestration. These results are illustrated in FIG. 7A and 7B. Views“a” and “b” show Fitc detection in cultures at 18 hours. Thatdistribution is exclusively nuclear for the nanoencapsules of RNAoligonucelotides (FIG. 7B: a vs. a′). Punctate inclusion are visible inthe cultures treated with the complexed RNA oligonucleotides thatco-localize with an immunological marker for lysosomes (FIG. 7A: a vs.a′). Particle sizing results from AFM microscopy are included todemonstrate dramatic difference in sizing following encapsulation. (FIG.7A, 7B:b vs.b, b′). Formulas encapsulating lower molecular weightdextrans and unstabilized RNA were also prepared with analagous uptake,nanocapsule size and yield to demonstrate that encapsulation can providenot only a targeting function but aid in stabilizing molecules sensitiveto chemical or enzymatic degradation. These examples demonstrates theusefulness of nanocapsules 36 for use in delivery of a broad range ofmacromolecules.

[0173] Although the present invention has been described with referenceto preferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1 5 1 6232 DNA Artificial Sequence Supplied by Invitrogen of Carlsbad,California 1 gtaccgaatt caagcttcgt gaggctccgg tgcccgtcag tgggcagagcgcacatcgcc 60 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcctagagaaggtg 120 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttcccgagggtgg 180 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgcaacgggtttgc 240 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctctttacgggtta 300 tggcccttgc gtgccttgaa ttacttccac ctggctccag tacgtgattcttgatcccga 360 gctggagcca ggggcgggcc ttgcgcttta ggagcccctt cgcctcgtgcttgagttgag 420 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcgcgcctgtctc 480 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgcgacgcttttt 540 ttctggcaag atagtcttgt aaatgcgggc caggatctgc acactggtatttcggttttt 600 gggcccgcgg ccggcgacgg ggcccgtgcg tcccagcgca catgttcggcgaggcggggc 660 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccggcctgctctg 720 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggctggcccggtcg 780 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctccagggggctcaaaa 840 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaaggaaaagggcc 900 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgccgtccaggcac 960 ctcgattagt tctggagctt ttggagtacg tcgtctttag gttggggggaggggttttat 1020 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagcttggcacttg 1080 atgtaattct ccttggaatt tggccttttt gagtttggat cttggttcattctcaagcct 1140 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaacacgtggccaccat 1200 ggcccaggtg cagctgcaga tggctagcaa aggagaagaa cttttcactggagttgtccc 1260 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtcagtggagaggg 1320 tgaaggtgat gctacatacg gaaagcttac ccttaaattt atttgcactactggaaaact 1380 acctgttcca tggccaacac ttgtcactac tttctcttat ggtgttcaatgcttttcccg 1440 ttatccggat catatgaaac ggcatgactt tttcaagagt gccatgcccgaaggttatgt 1500 acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtgctgaagtcaa 1560 gtttgaaggt gatacccttg ttaatcgtat cgagttaaaa ggtattgattttaaagaaga 1620 tggaaacatt ctcggacaca aactcgagta caactataac tcacacaatgtatacatcac 1680 ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccacaacattgaaga 1740 tggatccgtt caactagcag accattatca acaaaatact ccaattggcgatggccctgt 1800 ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaagatcccaacga 1860 aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggattacacatggcat 1920 ggatgagctc tacaaagcgg ccgcagatcc aaaaaagaag agaaaggtagatccaaaaaa 1980 gaagagaaag gtagatccaa aaaagaagag aaaggtagat acggccgcagaacaaaaact 2040 catctcagaa gaggatctga atggggccgc atagtctaga agctcgctgatcagcctcga 2100 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgccttccttgaccc 2160 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgcatcgcattgtc 2220 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaagggggaggatt 2280 gggaagacaa tagcaggcat gctggggatg gcccgggctc tatggcttctgaggcggaaa 2340 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgcattaagcgcgg 2400 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgccctagcgcccgctc 2460 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgtcaagctctaa 2520 atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgaccccaaaaaac 2580 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtttttcgccctt 2640 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactggaacaacactca 2700 accctatctc ggtctattct tttgatttat aagggatttt ggggatttcggcctattggt 2760 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtggaatgtgtgtca 2820 gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaaagcatgcatc 2880 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggcagaagtatgc 2940 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccgcccatcccgc 3000 ccctaactcc gcccagttcc gcccattctc cgcccctagg ctgactaattttttttattt 3060 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtgaggaggctttt 3120 ttggaggcct aggcttttgc aaaaagctcc cgggaggtcc acaatgattgaacaagatgg 3180 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatgactgggcaca 3240 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcaggggcgcccggt 3300 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctccaggacgaggcagcgcg 3360 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacgttgtcactga 3420 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcctgtcatctca 3480 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggctgcatacgct 3540 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagcgagcacgtac 3600 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatcaggggctcgc 3660 gccagccgaa ctgttcgcca ggctcaaggc gcgtatgccc gacggcgaggatctcgtcgt 3720 gactcatggc gatgcctgct tgccgaatat catggtggaa aatggccgcttttctggatt 3780 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgttggctacccg 3840 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgctttacggtat 3900 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagttcttctgagc 3960 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatcacgagatttc 4020 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccgggacgccggc 4080 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccccaacttgttt 4140 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcacaaataaagca 4200 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatcttatcatgtc 4260 tgtataccgg atctttccgc ttcctcgctc actgactcgc tgcgctcggtcgttcggctg 4320 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacagaatcaggggat 4380 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccgtaaaaaggcc 4440 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaaaaatcgacgc 4500 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtttccccctgga 4560 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacctgtccgccttt 4620 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatctcagttcggtg 4680 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcccgaccgctgc 4740 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgacttatcgccactg 4800 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgctacagagttc 4860 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtatctgcgctctg 4920 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaaacaaaccacc 4980 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaaaaaaggatct 5040 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacgaaaactcacgt 5100 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatccttttaaattaa 5160 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctgacagttaccaa 5220 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatccatagttgcc 5280 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctggccccagtgct 5340 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaataaaccagcca 5400 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccatccagtctatt 5460 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcgcaacgttgtt 5520 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttcattcagctcc 5580 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaaagcggttagc 5640 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatcactcatggtt 5700 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgcttttctgtgact 5760 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgagttgctcttgc 5820 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagtgctcatcatt 5880 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgagatccagttcg 5940 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcaccagcgtttct 6000 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggcgacacggaaa 6060 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatcagggttattgt 6120 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaataggggttccgcgc 6180 acatttcccc gaaaagtgcc acctgacgtc agatcgacgg atcgggagatcg 6232 2 2200 PRT Homo sapiens 2 Met Gly Ala Met Thr Gln Leu Leu AlaGly Val Phe Leu Ala Phe Leu 1 5 10 15 Ala Leu Ala Thr Glu Gly Gly ValLeu Lys Lys Val Ile Arg His Lys 20 25 30 Arg Gln Ser Gly Val Asn Ala ThrLeu Pro Glu Glu Asn Gln Pro Val 35 40 45 Val Phe Asn His Val Tyr Asn IleLys Leu Pro Val Gly Ser Gln Cys 50 55 60 Ser Val Asp Leu Glu Ser Ala SerGly Glu Lys Asp Leu Ala Pro Pro 65 70 75 80 Ser Glu Pro Ser Glu Ser PheGln Glu His Thr Val Asp Gly Glu Asn 85 90 95 Gln Ile Val Phe Thr His ArgIle Asn Ile Pro Arg Arg Ala Cys Gly 100 105 110 Cys Ala Ala Ala Pro AspVal Lys Glu Leu Leu Ser Arg Leu Glu Glu 115 120 125 Leu Glu Asn Leu ValSer Ser Leu Arg Glu Gln Cys Thr Ala Gly Ala 130 135 140 Gly Cys Cys LeuGln Pro Ala Thr Gly Arg Leu Asp Thr Arg Pro Phe 145 150 155 160 Cys SerGly Arg Gly Asn Phe Ser Thr Glu Gly Cys Gly Cys Val Cys 165 170 175 GluPro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro Glu Cys Pro Gly 180 185 190Asn Cys His Leu Arg Gly Arg Cys Ile Asp Gly Gln Cys Ile Cys Asp 195 200205 Asp Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu Ala Cys Pro Ser Asp 210215 220 Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val Cys Ile Cys Phe Glu225 230 235 240 Gly Tyr Ala Gly Ala Asp Cys Ser Arg Glu Ile Cys Pro ValPro Cys 245 250 255 Ser Glu Glu His Gly Thr Cys Val Asp Gly Leu Cys ValCys His Asp 260 265 270 Gly Phe Ala Gly Asp Asp Cys Asn Lys Pro Leu CysLeu Asn Asn Cys 275 280 285 Tyr Asn Arg Gly Arg Cys Val Glu Asn Glu CysVal Cys Asp Glu Gly 290 295 300 Phe Thr Gly Glu Asp Cys Ser Glu Leu IleCys Pro Asn Asp Cys Phe 305 310 315 320 Asp Arg Gly Arg Cys Ile Asn GlyThr Cys Tyr Cys Glu Glu Gly Phe 325 330 335 Thr Gly Glu Asp Cys Gly LysPro Thr Cys Pro His Ala Cys His Thr 340 345 350 Gln Gly Arg Cys Glu GluGly Gln Cys Val Cys Asp Glu Gly Phe Ala 355 360 365 Gly Leu Asp Cys SerGlu Lys Arg Cys Pro Ala Asp Cys His Asn Arg 370 375 380 Gly Arg Cys ValAsp Gly Arg Cys Glu Cys Asp Asp Gly Phe Thr Gly 385 390 395 400 Ala AspCys Gly Glu Leu Lys Cys Pro Asn Gly Cys Ser Gly His Gly 405 410 415 ArgCys Val Asn Gly Gln Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu 420 425 430Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp Cys His Ser Arg Gly Arg 435 440445 Cys Val Glu Gly Lys Cys Val Cys Glu Gln Gly Phe Lys Gly Tyr Asp 450455 460 Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His Gln His Gly Arg Cys465 470 475 480 Val Asn Gly Met Cys Val Cys Asp Asp Gly Tyr Thr Gly GluAsp Cys 485 490 495 Arg Asp Arg Gln Cys Pro Arg Asp Cys Ser Asn Arg GlyLeu Cys Val 500 505 510 Asp Gly Gln Cys Val Cys Glu Asp Gly Phe Thr GlyPro Asp Cys Ala 515 520 525 Glu Leu Ser Cys Pro Asn Asp Cys His Gly GlnGly Arg Cys Val Asn 530 535 540 Gly Gln Cys Val Cys His Glu Gly Phe MetGly Lys Asp Cys Lys Glu 545 550 555 560 Gln Arg Cys Pro Ser Asp Cys HisGly Gln Gly Arg Cys Val Asp Gly 565 570 575 Gln Cys Ile Cys His Glu GlyPhe Thr Gly Leu Asp Cys Gly Gln His 580 585 590 Ser Cys Pro Ser Asp CysAsn Asn Leu Gly Gln Cys Val Ser Gly Arg 595 600 605 Cys Ile Cys Asn GluGly Tyr Ser Gly Glu Asp Cys Ser Glu Val Ser 610 615 620 Pro Pro Lys AspLeu Val Val Thr Glu Val Thr Glu Glu Thr Val Asn 625 630 635 640 Leu AlaTrp Asp Asn Glu Met Arg Val Thr Glu Tyr Leu Val Val Tyr 645 650 655 ThrPro Thr His Glu Gly Gly Leu Glu Met Gln Phe Arg Val Pro Gly 660 665 670Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu Glu Pro Gly Val Glu Tyr 675 680685 Phe Ile Arg Val Phe Ala Ile Leu Glu Asn Lys Lys Ser Ile Pro Val 690695 700 Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly Leu Lys Phe705 710 715 720 Lys Ser Ile Lys Glu Thr Ser Val Glu Val Glu Trp Asp ProLeu Asp 725 730 735 Ile Ala Phe Glu Thr Trp Glu Ile Ile Phe Arg Asn MetAsn Lys Glu 740 745 750 Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg ProGlu Thr Ser Tyr 755 760 765 Arg Gln Thr Gly Leu Ala Pro Gly Gln Glu TyrGlu Ile Ser Leu His 770 775 780 Ile Val Lys Asn Asn Thr Arg Gly Pro GlyLeu Lys Arg Val Thr Thr 785 790 795 800 Thr Arg Leu Asp Ala Pro Ser GlnIle Glu Val Lys Asp Val Thr Asp 805 810 815 Thr Thr Ala Leu Ile Thr TrpPhe Lys Pro Leu Ala Glu Ile Asp Gly 820 825 830 Ile Glu Leu Thr Tyr GlyIle Lys Asp Val Pro Gly Asp Arg Thr Thr 835 840 845 Ile Asp Leu Thr GluAsp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys 850 855 860 Pro Asp Thr GluTyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met 865 870 875 880 Ser SerAsn Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu Asp Ala Pro 885 890 895 ArgAsn Leu Arg Arg Val Ser Gln Thr Asp Asn Ser Ile Thr Leu Glu 900 905 910Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr Arg Ile Lys Tyr Ala 915 920925 Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp Val Pro Lys Ser Gln 930935 940 Gln Ala Thr Thr Lys Thr Thr Leu Thr Gly Leu Arg Pro Gly Thr Glu945 950 955 960 Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp Lys Glu SerAsn Pro 965 970 975 Ala Thr Ile Asn Ala Ala Thr Glu Leu Asp Thr Pro LysAsp Leu Gln 980 985 990 Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu LeuTrp Lys Thr Pro 995 1000 1005 Leu Ala Lys Phe Asp Arg Tyr Arg Leu AsnTyr Ser Leu Pro Thr 1010 1015 1020 Gly Gln Trp Val Gly Val Gln Leu ProArg Asn Thr Thr Ser Tyr 1025 1030 1035 Val Leu Arg Gly Leu Glu Pro GlyGln Glu Tyr Asn Val Leu Leu 1040 1045 1050 Thr Ala Glu Lys Gly Arg HisLys Ser Lys Pro Ala Arg Val Lys 1055 1060 1065 Ala Ser Thr Glu Gln AlaPro Glu Leu Glu Asn Leu Thr Val Thr 1070 1075 1080 Glu Val Gly Trp AspGly Leu Arg Leu Asn Trp Thr Ala Ala Asp 1085 1090 1095 Gln Ala Tyr GluHis Phe Ile Ile Gln Val Gln Glu Ala Asn Lys 1100 1105 1110 Val Glu AlaAla Arg Asn Leu Thr Val Pro Gly Ser Leu Arg Ala 1115 1120 1125 Val AspIle Pro Gly Leu Lys Ala Ala Thr Pro Tyr Thr Val Ser 1130 1135 1140 IleTyr Gly Val Ile Gln Gly Tyr Arg Thr Pro Val Leu Ser Ala 1145 1150 1155Glu Ala Ser Thr Gly Glu Thr Pro Asn Leu Gly Glu Val Val Val 1160 11651170 Ala Glu Val Gly Trp Asp Ala Leu Lys Leu Asn Trp Thr Ala Pro 11751180 1185 Glu Gly Ala Tyr Glu Tyr Phe Phe Ile Gln Val Gln Glu Ala Asp1190 1195 1200 Thr Val Glu Ala Ala Gln Asn Leu Thr Val Pro Gly Gly LeuArg 1205 1210 1215 Ser Thr Asp Leu Pro Gly Leu Lys Ala Ala Thr His TyrThr Ile 1220 1225 1230 Thr Ile Arg Gly Val Thr Gln Asp Phe Ser Thr ThrPro Leu Ser 1235 1240 1245 Val Glu Val Leu Thr Glu Glu Val Pro Asp MetGly Asn Leu Thr 1250 1255 1260 Val Thr Glu Val Ser Trp Asp Ala Leu ArgLeu Asn Trp Thr Thr 1265 1270 1275 Pro Asp Gly Thr Tyr Asp Gln Phe ThrIle Gln Val Gln Glu Ala 1280 1285 1290 Asp Gln Val Glu Glu Ala His AsnLeu Thr Val Pro Gly Ser Leu 1295 1300 1305 Arg Ser Met Glu Ile Pro GlyLeu Arg Ala Gly Thr Pro Tyr Thr 1310 1315 1320 Val Thr Leu His Gly GluVal Arg Gly His Ser Thr Arg Pro Leu 1325 1330 1335 Ala Val Glu Val ValThr Glu Asp Leu Pro Gln Leu Gly Asp Leu 1340 1345 1350 Ala Val Ser GluVal Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr 1355 1360 1365 Ala Ala AspAsn Ala Tyr Glu His Phe Val Gln Val Gln Glu Val 1370 1375 1380 Asn LysVal Glu Ala Ala Gln Asn Leu Thr Leu Pro Gly Ser Leu 1385 1390 1395 ArgAla Val Asp Ile Pro Gly Leu Glu Ala Ala Thr Pro Tyr Arg 1400 1405 1410Val Ser Ile Tyr Gly Val Ile Arg Gly Tyr Arg Thr Pro Val Leu 1415 14201425 Ser Ala Glu Ala Ser Thr Ala Lys Glu Pro Glu Ile Gly Asn Leu 14301435 1440 Asn Val Ser Asp Ile Thr Pro Glu Ser Phe Asn Leu Ser Trp Met1445 1450 1455 Ala Thr Asp Gly Ile Phe Glu Thr Phe Thr Ile Glu Ile IleAsp 1460 1465 1470 Ser Asn Arg Leu Leu Glu Thr Val Glu Tyr Asn Ile SerGly Ala 1475 1480 1485 Glu Arg Thr Ala His Ile Ser Gly Leu Pro Pro SerThr Asp Phe 1490 1495 1500 Ile Val Tyr Leu Ser Gly Leu Ala Pro Ser IleArg Thr Lys Thr 1505 1510 1515 Ile Ser Ala Thr Ala Thr Thr Glu Ala LeuPro Leu Leu Glu Asn 1520 1525 1530 Leu Thr Ile Ser Asp Ile Asn Pro TyrGly Phe Thr Val Ser Trp 1535 1540 1545 Met Ala Ser Glu Asn Ala Phe AspSer Phe Leu Val Thr Val Val 1550 1555 1560 Asp Ser Gly Lys Leu Leu AspPro Gln Glu Phe Thr Leu Ser Gly 1565 1570 1575 Thr Gln Arg Lys Leu GluLeu Arg Gly Leu Ile Thr Gly Ile Gly 1580 1585 1590 Tyr Glu Val Met ValSer Gly Phe Thr Gln Gly His Gln Thr Lys 1595 1600 1605 Pro Leu Arg AlaGlu Ile Val Thr Glu Ala Glu Pro Glu Val Asp 1610 1615 1620 Asn Leu LeuVal Ser Asp Ala Thr Pro Asp Gly Phe Arg Leu Ser 1625 1630 1635 Trp ThrAla Asp Glu Gly Val Phe Asp Asn Phe Val Leu Lys Ile 1640 1645 1650 ArgAsp Thr Lys Lys Gln Ser Glu Pro Leu Glu Ile Thr Leu Leu 1655 1660 1665Ala Pro Glu Arg Thr Arg Asp Leu Thr Gly Leu Arg Glu Ala Thr 1670 16751680 Glu Tyr Glu Ile Glu Leu Tyr Gly Ile Ser Lys Gly Arg Arg Ser 16851690 1695 Gln Thr Val Ser Ala Ile Ala Thr Thr Ala Met Gly Ser Pro Lys1700 1705 1710 Glu Val Ile Phe Ser Asp Ile Thr Glu Asn Ser Ala Thr ValSer 1715 1720 1725 Trp Arg Ala Pro Thr Ala Gln Val Glu Ser Phe Arg IleThr Tyr 1730 1735 1740 Val Pro Ile Thr Gly Gly Thr Pro Ser Met Val ThrVal Asp Gly 1745 1750 1755 Thr Lys Thr Gln Thr Arg Leu Val Lys Leu IlePro Gly Val Glu 1760 1765 1770 Tyr Leu Val Ser Ile Ile Ala Met Lys GlyPhe Glu Glu Ser Glu 1775 1780 1785 Pro Val Ser Gly Ser Phe Thr Thr AlaLeu Asp Gly Pro Ser Gly 1790 1795 1800 Leu Val Thr Ala Asn Ile Thr AspSer Glu Ala Leu Ala Arg Trp 1805 1810 1815 Gln Pro Ala Ile Ala Thr ValAsp Ser Tyr Val Ile Ser Tyr Thr 1820 1825 1830 Gly Glu Lys Val Pro GluIle Thr Arg Thr Val Ser Gly Asn Thr 1835 1840 1845 Val Glu Tyr Ala LeuThr Asp Leu Glu Pro Ala Thr Glu Tyr Thr 1850 1855 1860 Leu Arg Ile PheAla Glu Lys Gly Pro Gln Lys Ser Ser Thr Ile 1865 1870 1875 Thr Ala LysPhe Thr Thr Asp Leu Asp Ser Pro Arg Asp Leu Thr 1880 1885 1890 Ala ThrGlu Val Gln Ser Glu Thr Ala Leu Leu Thr Trp Arg Pro 1895 1900 1905 ProArg Ala Ser Val Thr Gly Tyr Leu Leu Val Tyr Glu Ser Val 1910 1915 1920Asp Gly Thr Val Lys Glu Val Ile Val Gly Pro Asp Thr Thr Ser 1925 19301935 Tyr Ser Leu Ala Asp Leu Ser Pro Ser Thr His Tyr Thr Ala Lys 19401945 1950 Ile Gln Ala Leu Asn Gly Pro Leu Arg Ser Asn Met Ile Gln Thr1955 1960 1965 Ile Phe Thr Thr Ile Gly Leu Leu Tyr Pro Phe Pro Lys AspCys 1970 1975 1980 Ser Gln Ala Met Leu Asn Gly Asp Thr Thr Ser Gly LeuTyr Thr 1985 1990 1995 Ile Tyr Leu Asn Gly Asp Lys Ala Gln Ala Leu GluVal Phe Cys 2000 2005 2010 Asp Met Thr Ser Asp Gly Gly Gly Trp Ile ValPhe Leu Arg Arg 2015 2020 2025 Lys Asn Gly Arg Glu Asn Phe Tyr Gln AsnTrp Lys Ala Tyr Ala 2030 2035 2040 Ala Gly Phe Gly Asp Arg Arg Glu GluPhe Trp Leu Gly Leu Asp 2045 2050 2055 Asn Leu Asn Lys Ile Thr Ala GlnGly Gln Tyr Glu Leu Arg Val 2060 2065 2070 Asp Leu Arg Asp His Gly GluThr Ala Phe Ala Val Tyr Asp Lys 2075 2080 2085 Phe Ser Val Gly Asp AlaLys Thr Arg Tyr Lys Leu Lys Val Glu 2090 2095 2100 Gly Tyr Ser Gly ThrAla Gly Asp Ser Met Ala Tyr His Asn Gly 2105 2110 2115 Arg Ser Phe SerThr Phe Asp Lys Asp Thr Asp Ser Ala Ile Thr 2120 2125 2130 Asn Cys AlaLeu Ser Tyr Lys Gly Ala Phe Trp Tyr Arg Asn Cys 2135 2140 2145 His ArgVal Asn Leu Met Gly Arg Tyr Gly Asp Asn Asn His Ser 2150 2155 2160 GlnGly Val Asn Trp Phe His Trp Lys Gly His Glu His Ser Ile 2165 2170 2175Gln Phe Ala Glu Met Lys Leu Arg Pro Ser Asn Phe Arg Asn Leu 2180 21852190 Glu Gly Arg Arg Lys Arg Ala 2195 2200 3 8578 DNA ArtificialSequence Supplied by Invitrogen of Carlsbad, California 3 gacggatcgggagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagttaagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaatttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttaggcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgactagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccgcgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccattgacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtcaatgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgccaagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagtacatgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattaccatggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacggggatttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacgggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgtacggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactggcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaacttaagcttacca tggggggttc tcatcatcat catcatcatg gtatggctag 960 catgactggtggacagcaaa tgggtcggga tctgtacgac gatgacgata aggtacctaa 1020 ggatcagcttggagttgatc ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 1080 tacccaacttaatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 1140 ggcccgcaccgatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc 1200 ctggtttccggcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc 1260 cgatactgtcgtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac 1320 caacgtaacctatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg 1380 ttgttactcgctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat 1440 tatttttgatggcgttaact cggcgtttca tctgtggtgc aacgggcgct gggtcggtta 1500 cggccaggacagtcgtttgc cgtctgaatt tgacctgagc gcatttttac gcgccggaga 1560 aaaccgcctcgcggtgatgg tgctgcgttg gagtgacggc agttatctgg aagatcagga 1620 tatgtggcggatgagcggca ttttccgtga cgtctcgttg ctgcataaac cgactacaca 1680 aatcagcgatttccatgttg ccactcgctt taatgatgat ttcagccgcg ctgtactgga 1740 ggctgaagttcagatgtgcg gcgagttgcg tgactaccta cgggtaacag tttctttatg 1800 gcagggtgaaacgcaggtcg ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga 1860 gcgtggtggttatgccgatc gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg 1920 gagcgccgaaatcccgaatc tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac 1980 gctgattgaagcagaagcct gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct 2040 gctgctgctgaacggcaagc cgttgctgat tcgaggcgtt aaccgtcacg agcatcatcc 2100 tctgcatggtcaggtcatgg atgagcagac gatggtgcag gatatcctgc tgatgaagca 2160 gaacaactttaacgccgtgc gctgttcgca ttatccgaac catccgctgt ggtacacgct 2220 gtgcgaccgctacggcctgt atgtggtgga tgaagccaat attgaaaccc acggcatggt 2280 gccaatgaatcgtctgaccg atgatccgcg ctggctaccg gcgatgagcg aacgcgtaac 2340 gcgaatggtgcagcgcgatc gtaatcaccc gagtgtgatc atctggtcgc tggggaatga 2400 atcaggccacggcgctaatc acgacgcgct gtatcgctgg atcaaatctg tcgatccttc 2460 ccgcccggtgcagtatgaag gcggcggagc cgacaccacg gccaccgata ttatttgccc 2520 gatgtacgcgcgcgtggatg aagaccagcc cttcccggct gtgccgaaat ggtccatcaa 2580 aaaatggctttcgctacctg gagagacgcg cccgctgatc ctttgcgaat acgcccacgc 2640 gatgggtaacagtcttggcg gtttcgctaa atactggcag gcgtttcgtc agtatccccg 2700 tttacagggcggcttcgtct gggactgggt ggatcagtcg ctgattaaat atgatgaaaa 2760 cggcaacccgtggtcggctt acggcggtga ttttggcgat acgccgaacg atcgccagtt 2820 ctgtatgaacggtctggtct ttgccgaccg cacgccgcat ccagcgctga cggaagcaaa 2880 acaccagcagcagtttttcc agttccgttt atccgggcaa accatcgaag tgaccagcga 2940 atacctgttccgtcatagcg ataacgagct cctgcactgg atggtggcgc tggatggtaa 3000 gccgctggcaagcggtgaag tgcctctgga tgtcgctcca caaggtaaac agttgattga 3060 actgcctgaactaccgcagc cggagagcgc cgggcaactc tggctcacag tacgcgtagt 3120 gcaaccgaacgcgaccgcat ggtcagaagc cgggcacatc agcgcctggc agcagtggcg 3180 tctggcggaaaacctcagtg tgacgctccc cgccgcgtcc cacgccatcc cgcatctgac 3240 caccagcgaaatggattttt gcatcgagct gggtaataag cgttggcaat ttaaccgcca 3300 gtcaggctttctttcacaga tgtggattgg cgataaaaaa caactgctga cgccgctgcg 3360 cgatcagttcacccgtgcac cgctggataa cgacattggc gtaagtgaag cgacccgcat 3420 tgaccctaacgcctgggtcg aacgctggaa ggcggcgggc cattaccagg ccgaagcagc 3480 gttgttgcagtgcacggcag atacacttgc tgatgcggtg ctgattacga ccgctcacgc 3540 gtggcagcatcaggggaaaa ccttatttat cagccggaaa acctaccgga ttgatggtag 3600 tggtcaaatggcgattaccg ttgatgttga agtggcgagc gatacaccgc atccggcgcg 3660 gattggcctgaactgccagc tggcgcaggt agcagagcgg gtaaactggc tcggattagg 3720 gccgcaagaaaactatcccg accgccttac tgccgcctgt tttgaccgct gggatctgcc 3780 attgtcagacatgtataccc cgtacgtctt cccgagcgaa aacggtctgc gctgcgggac 3840 gcgcgaattgaattatggcc cacaccagtg gcgcggcgac ttccagttca acatcagccg 3900 ctacagtcaacagcaactga tggaaaccag ccatcgccat ctgctgcacg cggaagaagg 3960 cacatggctgaatatcgacg gtttccatat ggggattggt ggcgacgact cctggagccc 4020 gtcagtatcggcggagttcc agctgagcgc cggtcgctac cattaccagt tggtctggtg 4080 tcaaaaataataaagccgaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta 4140 gagggcccgtttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 4200 ttgtttgcccctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 4260 cctaataaaatgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 4320 gtggggtggggcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 4380 atgcggtgggctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc 4440 cccacgcgccctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4500 ccgctacacttgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4560 ccacgttcgccggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat 4620 ttagtgctttacggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4680 ggccatcgccctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4740 gtggactcttgttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4800 tataagggattttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4860 ttaacgcgaattaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc 4920 cccaggcaggcagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 4980 agtccccaggctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 5040 ccatagtcccgcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 5100 ctccgccccatggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 5160 ctgagctattccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 5220 tcccgggagcttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 5280 gcatgattgaacaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 5340 tcggctatgactgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 5400 cagcgcaggggcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 5460 tgcaggacgaggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 5520 tgctcgacgttgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 5580 aggatctcctgtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 5640 tgcggcggctgcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 5700 gcatcgagcgagcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 5760 aagagcatcaggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 5820 acggcgaggatctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 5880 atggccgcttttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 5940 acatagcgttggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 6000 tcctcgtgctttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 6060 ttgacgagttcttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 6120 cctgccatcacgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 6180 cgttttccgggacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 6240 cgcccaccccaacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 6300 aaatttcacaaataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 6360 caatgtatcttatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 6420 gtcatagctgtttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 6480 cggaagcataaagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 6540 gttgcgctcactgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 6600 cggccaacgcgcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 6660 tgactcgctgcgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 6720 aatacggttatccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 6780 gcaaaaggccaggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 6840 ccctgacgagcatcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 6900 ataaagataccaggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 6960 gccgcttaccggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 7020 ctcacgctgtaggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 7080 cgaaccccccgttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 7140 cccggtaagacacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 7200 gaggtatgtaggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 7260 aaggacagtatttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 7320 tagctcttgatccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 7380 gcagattacgcgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 7440 tgacgctcagtggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 7500 gatcttcacctagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 7560 tgagtaaacttggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 7620 ctgtctatttcgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 7680 ggagggcttaccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 7740 tccagatttatcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 7800 aactttatccgcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 7860 gccagttaatagtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 7920 gtcgtttggtatggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 7980 ccccatgttgtgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 8040 gttggccgcagtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 8100 gccatccgtaagatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 8160 gtgtatgcggcgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 8220 tagcagaactttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 8280 gatcttaccgctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 8340 agcatcttttactttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 8400 aaaaaagggaataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 8460 ttattgaagcatttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 8520 gaaaaataaacaaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 8578 4 4748 DNAArtificial Sequence Provided by Dr. Brett Levay-Young of the Universityof Minnesota 4 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatatatggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacccccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttccattgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgtatcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcattatgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtcatcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttgactcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcaccaaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcggtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatccgctagcgcta 600 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggtgcccatcctg 660 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcgagggcgagggc 720 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaagctgcccgtg 780 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcagccgctacccc 840 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggctacgtccaggag 900 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggtgaagttcgag 960 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaaggaggacggcaac 1020 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatatcatggccgac 1080 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcgaggacggcagc 1140 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccccgtgctgctg 1200 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaacgagaagcgc 1260 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcggcatggacgag 1320 ctgtacaagt actcagatct cgagctcaag cttaaccctc cggacgagagcggccctggc 1380 tgtatgtcct gcaagtgcgt gctgtcctga tcaccggatc tagataactgatcataatca 1440 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacacctccccctga 1500 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgcagcttataatg 1560 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttttcactgcatt 1620 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgtaagcgttaat 1680 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaaccaataggcc 1740 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggttgagtgttgtt 1800 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaaagggcgaaaa 1860 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaagttttttgggg 1920 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatttagagcttga 1980 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaaggagcgggcgct 2040 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgccgcgcttaat 2100 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcggaacccctatt 2160 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaataaccctgataa 2220 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccagctgtggaatg 2280 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagtatgcaaagca 2340 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctccccagcaggcagaa 2400 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgcccctaactccgccca 2460 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctgactaatttttt 2520 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaagtagtgaggag 2580 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatgaggatcgttt 2640 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggtggagaggcta 2700 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgtgttccggctg 2760 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgccctgaatgaa 2820 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttccttgcgcagct 2880 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcgaagtgccgggg 2940 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcatggctgatgca 3000 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccaccaagcgaaacat 3060 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcaggatgatctggac 3120 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggcgagcatgccc 3180 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatatcatggtggaa 3240 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcggaccgctatcag 3300 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatgggctgaccgc 3360 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgccttctatcgcctt 3420 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaagcgacgccca 3480 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttgggcttcggaa 3540 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatgctggagttct 3600 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccggaaggaaccc 3660 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcgtttgttcata 3720 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgagaccccattggg 3780 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcgggtgaaggcc 3840 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggttactcatata 3900 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtgaagatccttt 3960 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactgagcgtcagacc 4020 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgtaatctgctgct 4080 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaagagctaccaa 4140 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatactgtccttctag 4200 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctacatacctcgctc 4260 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtcttaccgggttgg 4320 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacggggggttcgtgca 4380 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacagcgtgagctat 4440 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggtaagcggcaggg 4500 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtatctttatagtc 4560 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcgtcaggggggc 4620 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggccttttgctggc 4680 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataaccgtattaccg 4740 ccatgcat 4748 5 4992 DNA Artificial Sequence Supplied byBD Biosciences Clonetech of Palo Alto, California 5 tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggagtatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgaccttatgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggaggtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggtcgccaccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 660 gtcgagctggacggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 720 gatgccacctacggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 780 ccctggcccaccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 840 gaccacatgaagcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 900 cgcaccatcttcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 960 ggcgacaccctggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1020 atcctggggcacaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1080 aagcagaagaacggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1140 gtgcagctcgccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1200 cccgacaaccactacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1260 gatcacatggtcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1320 ctgtacaagtactcagatct cgagctcaag cttaccatgg ggggttctca tcatcatcat 1380 catcatggtatggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat 1440 gacgataaggggactgctgc ggccaatgcg aacgacttct tcgccaagcg caagagaact 1500 gcgcaggagaacaaggcgtc gaacgacgtc cctccagggt gtccctctcc aaacgtggct 1560 cctggggtgggcgcggtgga gcagaccccg cgcaaacgtc tgagatgagg atccagtgtg 1620 gtggaattctgcagatatcc agcacagtgg cggccgctcg agtctagata actgatcata 1680 atcagccataccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 1740 ctgaacctgaaacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 1800 aatggttacaaataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 1860 cattctagttgtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt 1920 taatattttgttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1980 ggccgaaatcggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 2040 tgttccagtttggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 2100 aaaaaccgtctatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 2160 ggggtcgaggtgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 2220 ttgacggggaaagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 2280 cgctagggcgctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 2340 taatgcgccgctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2400 tatttgtttatttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 2460 ataaatgcttcaataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg 2520 aatgtgtgtcagttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2580 agcatgcatctcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 2640 agaagtatgcaaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 2700 cccatcccgcccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 2760 ttttttatttatgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 2820 ggaggcttttttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc 2880 gtttcgcatgattgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 2940 gctattcggctatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3000 gctgtcagcgcaggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3060 tgaactgcaagacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3120 agctgtgctcgacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3180 ggggcaggatctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3240 tgcaatgcggcggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3300 acatcgcatcgagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3360 ggacgaagagcatcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 3420 gcccgacggcgaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3480 ggaaaatggccgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3540 tcaggacatagcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3600 ccgcttcctcgtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 3660 ccttcttgacgagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 3720 cccaacctgccatcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3780 ggaatcgttttccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 3840 ttcttcgcccaccctagggg gaggctaact gaaacacgga aggagacaat accggaagga 3900 acccgcgctatgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt 3960 cataaacgcggggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat 4020 tggggccaatacgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa 4080 ggcccagggctcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca 4140 tatatactttagattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 4200 ctttttgataatctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 4260 gaccccgtagaaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 4320 tgcttgcaaacaaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4380 ccaactctttttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 4440 ctagtgtagccgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 4500 gctctgctaatcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4560 ttggactcaagacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4620 tgcacacagcccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4680 ctatgagaaagcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4740 agggtcggaacaggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4800 agtcctgtcgggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4860 gggcggagcctatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4920 tggccttttgctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4980 accgccatgcat 4992

1. A composition comprising: a plurality of particles comprising asurfactant having an HLB value of less than about 6.0 units associatedwith a functional composition and a polymer, with the particles havingan average diameter of less than about 100 nanometers as measured byatomic force microscopy of the particles following drying of theparticles, wherein the functional composition is a member of the groupconsisting of a bioactive component and a diagnostic agent.
 2. Thecomposition of claim 1 wherein the surfactant is a non-ionic surfactant.3. The composition of claim 1 wherein the surfactant has an HLB value ofless than about 5.0 units.
 4. The composition of claim 3 wherein theaverage diameter of the particles is less than about 50 nm.
 5. Thecomposition of claim 1 wherein the functional composition is a member ofthe group consisting of peptides, proteins, and carbohydrates.
 6. Thecomposition of claim 5 wherein the average diameter is less than about50 nm.
 7. The composition of claim 1 wherein the functional compositionis cisplatin.
 8. The composition of claim 1 wherein the functionalcomposition is inorganic.
 9. The composition of claim 1 wherein thefunctional composition is a diagnostic agent that is a fluorescentmolecule.
 10. The composition of claim 1 wherein the functionalcomposition is a bioactive component that comprises a hydrophiliccomponent.
 11. The composition of claim 10 wherein the bioactivecomponent is condensed.
 12. The composition of claim 1 wherein thefunctional composition is a bioactive component that is a member of thegroup consisting of aptamers, mini-chromosomes, steroids, adrenergic,adrenocotical steroid, adrenocortical suppressant, aldosteroneantagonist, and anabolic agents; analeptic, analgesic, anesthetic,anorectic, anti-acne agents; anti-adrenergic, anti-allergic,anti-amebic, anti-anemic, and anti-anginal agents; anti-arthritic,anti-asthmatic, anti-atherosclerotic, antibacterial, and anticholinergicagents; anticoagulant, anticonvulsant, antidepressant, antidiabetic, andantidiarrheal agents; antidiuretic, anti-emetic, anti-epileptic,antifibrinolytic, and antifungal agent; antihemorrhagic, inflammatory,antimicrobial, antimigraine, and antimiotic agents; antimycotic,antinauseant, antineoplastic, antineutropenic, and antiparasitic agents;antiproliferative, antipsychotic, antirheumatic, antiseborrheic, andantisecretory agents; antipasmodic, antihrombotic, anti-ulcerative,antiviral and appetite suppressant agents.
 13. The composition of claim1 wherein the functional composition is a bioactive component that is amember of the group consisting of blood glucose regulator, boneresorption inhibitor, bronchodilator, cardiovascular, and cholinergicagents; fluorescent, free oxygen radical scavenger, gastrointestinalmotility effector, glucocorticoid, and hair growth stimulant agent;hemostatic, histamine H₂ receptor antagonists; hormone;hypocholesterolemic, and hypoglycemic agents; hypolipidemic,hypotensive, and imaging agents, immunizing and agonist agents; moodregulators, mucolytic, mydriatic, nasal decongestant; neuromuscularblocking agents; neuroprotective, NMDA antagonist, non-hormonal sterolderivative, plasminogen activator, and platelet activating factorantagonist agent.
 14. The composition of claim 1 wherein the functionalcomposition is a bioactive component that is a member of the groupconsisting of platelet aggregation inhibitor, psychotropic, radioactive,scabicide, and sclerosing agents; sedative, sedative-hypnotic, selectiveadenosine A1 antagonist, serotonin antagonist, and serotonin inhibitoragent; serotonin receptor antagonist, steroid, thyroid hormone, thyroidhormone, thyroid inhibitor agent; thyromimetic, tranquilizer,amyotrophic lateral sclerosis, cerebral ischemia, Pagel's disease agent;unstable angina, vasoconstrictor, vasodilator, wound healing, andxanthine oxidase inhibitor agent; and immunological agents.
 15. Thecomposition of claim 1 wherein the functional composition is a bioactivecomponent that comprises a member of the group consisting of antigensisolated from pathogens, viral antigens, fungal antigens, parasiticantigens, and inactivated pathogenic organisms.
 16. The composition ofclaim 1 wherein the surfactant has a critical micelle concentration ofless than about 200 micromolar.
 17. The composition of claim 1 furthercomprising a biocompatible oil.
 18. The composition of claim 1 whereinthe functional composition is a bioactive component that comprises apolynucleic acid, oligonucleotide, antisense molecule, or a polypeptide.19. The composition of claim 18 wherein the bioactive component iscondensed.
 20. The composition of claim 19 wherein the compositionfurther comprises a water-miscible solvent.
 21. The composition of claim1 wherein the surfactant is selected from the group consisting of 2, 4,7, 9-tetramethyl-5-decyn-4, 7-diol, molecules containing an acetylenicdiol portion, and blends of 2, 4, 7, 9-tetramethyl-5-decyn-4, 7-diol.22. The composition of claim 1 wherein the particles further comprise acell recognition component.
 23. The composition of claim 22 wherein thecell recognition component is a ligand, peptide hormone, or an antibody.24. The composition of claim 23 wherein the cell recognition componentcomprises tenascin, hyaluronan, or polyvinylpyrrolidone, or a fragmentthereof.
 25. The composition of claim 24 wherein the average diameter ofthe particles is less than about 50 nm.
 26. The composition of claim 24wherein the cell recognition component comprises tenascin or a fragmentthereof.
 27. The composition of claim 1 wherein the average diameter ofthe particles is less than about 50 nm.
 28. The composition of claim 1wherein the polymer is an iontophoretic polymer.
 29. The composition ofclaim 1 wherein the polymer is a hydrophobic polymer.
 30. Thecomposition of claim 1 wherein the polymer is a hydrophilic polymer. 31.The composition of claim 1 wherein the polymer is chosen from the groupconsisting of polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andcopolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt.
 32. Thecomposition of claim 1 wherein the polymer is chosen from the groupconsisting of poly(methyl methacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene poly(ethylene glycol),poly(ethylene oxide), and poly(ethylene terephthalate).
 33. Thecomposition of claim 1 wherein the polymer is chosen from the groupconsisting of poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene, polyvinylpryrrolidone, polyhyaluronic acids,casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexlmethacrylate), poly(isodecl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadeclacrylate).
 34. The composition of claim 1 wherein the hydrophilicpolymer is a member of the group consisting of proteinaceous materials ,peptides, carbohydrates.
 35. A method of delivering a functionalcomposition across keratinized barrier epithelia to a cell, the methodcomprising introducing the composition of claim 1 at a position that isseparated from the cell by a keratinized barrier epithelium, wherein atleast a portion of the plurality of particles passes through thekeratinized barrier epithelium to the cell.
 36. The method of claim 35wherein the functional composition is delivered transcutaneously. 37.The method of claim 35 wherein the composition of claim 1 is prepared asa medicament, and the medicament is administered to a patient.
 38. Asolution comprising the composition of claim 1, the solution comprisinga concentration of cations between 20 and 2000 millimolar.
 39. Thecomposition of claim 1, further comprising a cation chosen from thegroup consisting of Ni²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Al³⁺, Be²⁺, Li⁺, Ba²⁺, andGd³⁺.
 40. A medicament comprising the composition of claim
 1. 41. Themedicament of claim 39 further comprising a form selected from the groupconsisting of granules, tablets, pellets, films, oral, intravenous,subcutaneous, intraperitoneal, intrathecal, intramuscular, inhalation,topical, transdermal, suppository, pessary, intra urethral, intraportal,intraocular, transtympanic, intrahepatic, intra-arterial, intrathecal,transmucosal, coatings, buccal, and combinations thereof.
 42. A methodof delivering a functional composition to a patient, the methodcomprising administering a medicament to the patient that comprises thecomposition of claim
 1. 43. A cell comprising the composition of claim1, wherein the plurality of particles is associated with the cell.
 44. Acell comprising the composition of claim 18, wherein the plurality ofparticles is associated with the cell.
 45. A method of transfecting acell, the method comprising exposing the cell to the composition ofclaim
 18. 46. A matrix for binding bioactive or diagnostic particles,the matrix comprising the particles of claim 1 and a binder.
 47. Amethod of delivering a medical agent to a cell having caveolae, themethod comprising: associating the medical agent with an organicfunctional composition in vitro to make an association of the medicalagent and the organic functional composition, wherein the assoication ispassable through cellular caveolae for delivery of the medical agent.48. The method of claim 47 wherein the association of the agent and thefunctional composition comprises a particle.
 49. The method of claim 48wherein the particle has a diameter of less than about 100 nanometers asmeasured by atomic force microscopy of the particles following drying ofthe particles.
 50. The method of claim 48 wherein the particle has adiameter of less than about 50 nm.
 51. The method of claim 48 whereinthe particle further comprises a surfactant having an HLB value of lessthan about 6.0 units.
 52. The method of claim 51 wherein the particlehas an average diameter of less than about 50 nanometers as measured byatomic force microscopy of the particles following drying of theparticles.
 53. The method of claim 51 further comprising exposing theparticle to the cell.
 54. The method of claim 47 wherein the associationof the agent and the functional composition has a maximum dimension ofno more than about 50 nm nanometers as measured by atomic forcemicroscopy following drying of the association of the agent and thefunctional composition.
 55. The method of claim 47 wherein thefunctional composition comprises a surfactant.
 56. The method of claim47 further comprising exposing the association of the agent and thefunctional composition to the cell.
 57. The method of claim 47 furthercomprising administering a medicament to a patient, the medicamentcomprising the association of the medical agent and the organicfunctional composition.
 58. The method of claim 47 wherein thefunctional composition comprises a surfactant and a hydrophilic polymer.59. The method of claim 47 wherein the agent comprises a bioactivecomponent that a member of the group consisting of peptides, proteins,and carbohydrates.
 60. The method of claim 47 wherein the agentcomprises a member of the group consisting of a bioactive component anda diagnostic agent.
 61. The method of claim 47 wherein the agentcomprises a fragment of a nucleic acid that comprises a nucleic acidsequence.
 62. The method of claim 61 wherein the functional compositioncomprises a surfactant.
 63. The method of claim 47 wherein theassociation is introduced at a position that is separated from the cellby keratinized barrier epithelia, and the association passes through thekeratinized barrier epithelia to the cell.
 64. The method of claim 63further comprising exposing the cell to the association of the agent andthe functional composition
 65. The method of claim 47 wherein thefunctional composition comprises carbon and hydrogen.